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Molecular and Cellular Biology, July 2000, p. 5175-5183, Vol. 20, No. 14
Department of Genetics, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania
19104-61451; Institut de
Génétique et de Biologie Moléculaire et Cellulaire,
CNRS/INSERM/Université Louis Pasteur, 67404 Illkirch Cedex,
France2; and Department of Molecular
Physiology and Biophysics, Vanderbilt University School of
Medicine, Nashville, Tennessee 372323
Received 21 December 1999/Returned for modification 3 February
2000/Accepted 5 April 2000
Liver-specific gene expression is controlled by a heterogeneous
group of hepatocyte-enriched transcription factors. One of these, the
winged helix transcription factor hepatocyte nuclear factor 3 Hepatic gene expression is
controlled by a diverse set of transcription factors, none of which is
expressed exclusively in the liver. Among these are the hepatocyte
nuclear factor 3 Recently, the nomenclature of the winged helix/forkhead transcription
factor gene family has been revised, and according to this new
nomenclature the genetic loci encoding HNF3 Analysis of the crystal structure of the DNA binding domain of HNF3 During formation of the definite endoderm, HNF3 In contrast to the HNF3 Recently, the regulatory function of HNF3 Generation of HNF3
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Hepatocyte Nuclear Factor 3
(Foxa2) Is Dispensable for
Maintaining the Differentiated State of the Adult Hepatocyte
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
(HNF3 or Foxa2) is essential for multiple
stages of embryonic development. Recently, HNF3 has been
shown to be an important regulator of other hepatocyte-enriched
transcription factors as well as the expression of liver-specific
structural genes. We have addressed the role of HNF3 in
maintenance of the hepatocyte phenotype by inactivation of
HNF3 in the liver. Remarkably, adult mice lacking
HNF3 expression specifically in hepatocytes are viable,
with histologically normal livers and normal liver function. Moreover,
analysis of >8,000 mRNAs by array hybridization revealed that lack of
HNF3 affects the expression of only very few genes.
Based on earlier work it appears that HNF3 plays a critical role in early liver development; however, our studies demonstrate that HNF3 is not required for maintenance of
the adult hepatocyte or for normal liver function. This is the first example of such functional dichotomy for a tissue specification transcription factor.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
, , and 
proteins (HNF3, HNF3, and
HNF3, respectively), which were first discovered by their ability to
bind to the promoters of the liver-specific genes encoding
1-antitrypsin and transthyretin (8, 20, 21). The
HNF3 genes are closely related to the Drosophila
melanogaster gene forkhead, which is essential for the
proper formation of the foregut and hindgut (32). This fact together with the observation that the mouse HNF3 genes are
expressed early during the formation of definite endoderm led to the
hypothesis that the HNF3 proteins function in mammalian liver and gut
development (3, 23, 27).
, HNF3, and HNF3
are now known as Foxa1, Foxa2, and
Foxa3, respectively, in mouse (Fox refers to
forkhead box) (17).
showed a striking similarity to the winged helix domain of linker
histones (7). The HNF3 proteins can bind to and reposition nucleosomes in the albumin gene which correlates with an active albumin
enhancer (6, 22, 29). As the HNF3 proteins can alter
chromatin and are the earliest known factors expressed in the
prehepatic endoderm, the HNF3 proteins were proposed to act as genetic
potentiators of the hepatic differentiation program (34).
is
activated first, followed by HNF3, and finally
HNF3 (3, 23, 27). HNF3 is
required for the development of the node and for visceral endoderm
formation because these structures are missing or abnormal in embryos
homozygous for a targeted null mutation in the HNF3 gene
(2, 10, 33). However, the functions of HNF3 in
the differentiation of the hepatic primordium or in hepatic metabolism could not be addressed using the HNF3 null allele, as
even embryos obtained from tetraploid embryonic stem (ES) cell
aggregations lacked foregut and midgut endoderm (10).
/ mice, embryos
deficient for HNF3 or HNF3 develop normally
to term and show no obvious morphological liver phenotype (15, 16,
28). HNF3 mutant mice had unchanged expression of
liver-specific HNF3 target genes involved in metabolism and glucose
homeostasis. However, the transcription of several HNF3 targets was
reduced by 50 to 70% in the HNF3 mutants. Due to the
similarity of the HNF3 proteins it seems likely that these transcription factors can at least partially compensate for each other
during liver development, when all three genes are expressed concurrently.
has been
studied in visceral endoderm derived from
HNF3/ embryoid bodies in vitro
(12). In this model, the lack of HNF3 resulted
in a reduction of the mRNA levels for the transcription factors
HNF1 and HNF4 and a complete elimination of
the HNF3 transcript, suggesting that HNF3
regulates a transcription factor network required for differentiation
and metabolism (12). To test the role of HNF3
in regulating the hepatic transcriptional program in vivo, we have
generated mice lacking HNF3 specifically in hepatocytes
using the Cre-loxP recombination system. Here we describe the
liver-specific gene expression profile and metabolic regulation in
these hepatocyte-specific HNF3 knockout mice.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
loxP/loxP Alb.Cre mice.
Lambda phage clones containing the murine HNF3 gene had been
isolated from a mouse ES cell (strain 129) library previously (2). The targeting vector is depicted in Fig.
1A and contains approximately 5 kb of
homology regions. This targeting construct was electroporated into
E14/1 mouse ES cells (18). Stably transfected cells were
isolated after selection in G418 (350 µg/ml; Gibco), and 340 clones
were screened for the desired homologous recombination event. A 0.8-kb
XhoI/BglII fragment (3' probe in Fig. 1A) was used as an external probe for Southern blot analysis of DNA digested with BglII (data not shown). The neo-tk cassette
was then removed from correctly targeted clones by partial Cre-mediated
recombination. Two clones were expanded and transfected transiently
with 2 µg of Cre expression vector (pIC-Cre [13]).
Two days posttransfection, cells were treated with 2 µM gancyclovir
to select for the cells that had lost the neo-tk cassette.
Ninety-six individual gancyclovir-resistant clones were analyzed by
Southern blotting (data not shown). Approximately 20% of the clones
were identified to have undergone partial recombination to generate the
HNF3loxP allele, thereby removing the neo-tk
cassette and one loxP site but leaving both exon 3 and its flanking
loxP sites intact. Two of the HNF3loxP ES cell clones
were injected into C57BL/6J mouse blastocysts. Blastocysts were
transferred to pseudopregnant NMRI females, and chimeric offspring were
identified by the presence of agouti hair. Chimeric males were mated to
C57BL/6 females to obtain ES cell-derived offspring that were analyzed
by Southern blotting of tail DNA to identify the
HNF3loxP/+ heterozygote mice. Germ line chimeras were
crossed to CD1 outbred mice, as this is the strain of mice used
previously for the analysis of the HNF3 null allele
(2). Heterozygotes were mated inter se to generate
homozygous HNF3loxP/loxP mice. The derivation of the
Alb.Cre transgenic line has been described previously (25),
and mice were kept on a CD1 background.
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FIG. 1.
Cre-loxP-mediated targeting of the HNF3
gene and generation of homozygous HNF3loxP/loxP mice.
(A) Targeting vector for the HNF3loxP allele.
Primers 5' and 3' were used in PCR genotype analysis. (First
line) Gene structure of the endogenous HNF3 locus. Exons
are indicated as boxes, the striped box represents the winged helix
domain, open triangles represent loxP sites, and arrow heads represent
primer positions. (Second line) Targeting vector which introduces a
cassette containing the neomycin-herpes simplex virus-thymidine kinase
selection cassette (neo-tk) flanked by loxP sites downstream of exon 3. An additional loxP site was introduced in the intron upstream of exon
3. (Third line) Gene structure of homologous recombinants. The 3' probe
was used for the Southern blot analysis (data not shown). (Fourth line)
Cre-mediated deletion results in either the HNF3 null
allele (deletion of exon 3) or the HNF3loxP
allele (exon 3 flanked by loxP sites). Abbreviations: Bg,
BglII; E, EcoRI; Xh, XhoI; Xba,
XbaI. (B) HNF3loxP/loxP mice are viable and
healthy. Shown are the results of PCR genotype analysis of a litter
from mating HNF3loxP/+ heterozygotes inter se. The
HNF3loxP allele segregates in expected
Mendelian frequency. WT, wild type.
Genotype analysis.
The genotypes of all offspring were
analyzed using DNA isolated either from the yolk sac of embryos or the
tails of 4-week-old mice. The 5' and 3' primers for the
Alb.Cre transgene (PCR product of 232 bp) were
5'-GCGGCATGGTGCAAGTTGAAT-3' and
5'-CGTTCACCGGCATCAACGTTT-3', respectively. The 5' and 3'
primers for the HNF3 gene whose positions are depicted in
Fig. 1a were 5'-CCCCTGAGTTGGCGGTGGT-3' and
5'-TTGCTCACGGAAGAGTAGCC-3', respectively, which
produce an ~290-bp amplified fragment for the wild-type
HNF3 allele and an ~450-bp amplified fragment for the
HNF3loxP allele. PCRs were carried out for 32 cycles (95°C for 45 s, 60°C for 45 s; 72°C for 90 s) in a buffer containing 1.5 mM MgCl2.
Immunohistochemistry.
Adult and fetal tissue samples were
fixed in 4% paraformaldehyde overnight at 4°C, embedded in paraffin,
cut to 6-µm-thick sections and applied to Probe-on Plus slides
(Fisher Scientific). Deparaffinized and rehydrated slides were
subjected to microwave antigen retrieval by boiling for 6 min in a 10 mM citric acid buffer, pH 6.0, and allowed to cool for 10 min at room
temperature (RT). Slides were washed in phosphate-buffered saline
(PBS), then blocked with avidin and biotin blocking agent (Vector) for
15 min each at RT, and then blocked with protein blocking reagent (Immunotech) for 20 min at RT. The primary antibody, a 1:10,000 dilution of polyclonal rabbit anti-HNF-3, kindly provided by Thomas
M. Jessell (Columbia University, New York, N.Y.), was diluted in PBS
containing 0.1% bovine serum albumin (BSA) and 0.2% Triton X-100
(PBT) and incubated with the sections overnight at 4°C. Slides were
washed in PBS, then incubated with goat anti-rabbit biotinylated
secondary antibody (Biomeda Biostain Rabbit IgG [AP] kit) diluted in
PBT for 30 min at 37°C, washed in PBS, and incubated with alkaline
phosphatase-conjugated ABC reagent (Biomeda) diluted in PBT for 30 min
at 37°C. Slides were washed in PBS and then washed in a solution
containing 100 mM NaCl, 50 mM MgCl2, 100 mM Tris (pH 9.5),
and 0.1% Tween 20. Signal was developed in Nitro Blue Tetrazolium
(0.35 mg/ml; Boehringer Mannheim), BCIP
(5-bromo-4-chloro-3-indolylphosphate (0.18 mg/ml; Boehringer Mannheim)
for 8 min at RT. Slides were washed in water, counterstained in a 2%
neutral red solution for 20 min, dehydrated, mounted, and viewed using
Nomarski optics on a Nikon X4Z microscope.
RNA analysis and liver function tests. Northern blot, RNase protection, and reverse transcription-PCR analysis were performed as described previously (12, 16). The primers used for reverse-transcription-PCR are available upon request. Microarray hybridizations were carried out by Incyte Pharmaceuticals, Inc. (Palo Alto, Calif.). For liver function tests, mice were fasted for 9 h before sacrifice by carbon dioxide asphyxiation. Whole venous blood obtained from the inferior vena cava was mixed with an anticoagulant consisting of trasylol, EDTA, and leupeptin and centrifuged for 5 min at 14,000 × g to obtain plasma. Plasma parameters were determined by Ani Lytics, Inc. (Gaithersburg, Md.).
Preparation of nuclear extracts from liver.
Livers from
16-week-old adult mice were extracted, rinsed in cold PBS, minced, and
dounced in homogenization buffer (2 M sucrose, 10 mM HEPES (pH 7.6), 25 mM KCl, 1 mM EDTA, 0.15 mM spermine, 0.5 mM spermidine, 0.5 mM
phenylmethylsulfonyl fluoride [PMSF], 0.5 mM dithiothreitol [DTT],
and 10% glycerol) until most of the cells were disrupted but nuclei
were still intact. The liver homogenates were then layered on top of
homogenization buffer in an ultracentrifuge tube and centrifuged at
27,000 rpm (90,000 × g) for 55 min at 4°C. The
nuclear pellet was resuspended in 0.1 ml of nuclear storage buffer (20 mM Tris [pH 7.9], 75 mM NaCl, 0.5 mM EDTA, 0.125 mM PMSF, 0.85 mM
DTT, and 50% glycerol). A 0.01-ml aliquot of the nuclear pellet was
used to normalize nuclear extract concentration as follows: 0.2 ml of
1% sodium dodecyl sulfate was added to the nuclear pellet aliquot,
vortexed, and centrifuged at 15,000 × g for 3 min.
Absorbance at 260 nm was assayed to estimate the amount of nucleic acid
in the nuclear extract. The remainder of the nuclear extract was
centrifuged at 3,000 rpm for 15 min at 4°C. The nuclei were
resuspended in nuclear storage buffer at 25 µg/ml. Nine volumes of
1.1× NUN solution (27.5 mM HEPES [pH 7.6], 1.1 M urea, 0.33 M NaCl,
1.1 mM DTT, and 1.1% NP-40) was added, vortexed briefly, incubated on
ice for 15 min, and centrifuged at 14,000 rpm at 4°C. Glycerol was
added to the supernatant containing the nuclear extract to a 10% final
volume and the solution was dialyzed against a 1,000× volume of Gorski
dialysis buffer (25 mM HEPES [pH 7.9], 40 mM KCl, 0.1 mM EDTA, 0.5 mM
PMSF, 1 mM DTT, and 10% glycerol) for 2 h at 4°C. Samples were
subsequently centrifuged at 14,000 rpm for 1 min at 4°C, aliquoted,
and stored at 
80°C. Additionally, all buffers contained the
following protease inhibitors: 0.1 mM benzamidine; 1 µg of antipain,
leupeptin, and soybean trypsin inhibitor per ml; and 2 µg of
aprotinin and bestatin per ml.
Electrophoretic mobility shift assays (EMSAs). The binding reaction contained 2.0 µg of nuclear extract in a 15-µl solution of 10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, and 1% Ficoll, with 200 ng of poly(dI-dC) and 2 µg of bovine serum albumin. The mixtures were incubated at RT for 10 min, and then an 80-fold molar excess of competitor oligonucleotide (as indicated) and 0.1 ng (5 × 104 to 10 × 104 cpm) of oligonucleotide probe were added to the reaction mixture. Incubation was continued for an additional 20 min prior to electrophoresis on 8% polyacrylamide gels in 1× Tris-borate-EDTA, and gels were dried and exposed to a PhosphorImager cassette. Oligonucleotide probes were labeled with [32P]dCTP by filling in the ends with Klenow polymerase I. The sequences of the double-stranded oligonucleotides used as probes or competitors have been described previously (9).
Glucose tolerance test. Overnight-fasted 8- to 15-week-old mice were injected intraperitoneally with 5 mg of glucose per g of body weight. Blood samples were obtained from the tail vein, and glucose levels were measured immediately before and 30, 60, and 120 min after injection using a Glucometer Elite device (Bayer, Inc.).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Generation of HNF3loxP mice.
We
have employed conditional gene inactivation using Cre-loxP-mediated
recombination to study the role of HNF3 in the liver, as embryos
homozygous for a HNF3 null allele die before hepatic differentiation begins (13, 25, 31). To achieve this, we constructed a targeting vector that introduced two loxP sites flanking
exon 3 of the HNF3 gene, which contains the bulk of the
protein-coding sequence, including the DNA binding domain (14), and a third loxP site downstream of a selection
cassette consisting of the neomycin resistance and herpes simplex virus thymidine kinase genes (neo-tk) (Fig. 1A). We chose exon 3 because deletion of this exon in the germ line resulted in complete
inactivation of the HNF3 gene (2, 33). After
electroporation of mouse ES cells, screening of 340 neomycin
(G418)-resistant colonies yielded three correctly targeted clones (data
not shown). As the presence of the neo-tk cassette might
interfere with the promoter activity and/or mRNA processing of the
HNF3 gene, this neo-tk cassette was
subsequently deleted in two of the targeted clones by transient
transfection with a Cre expression vector. As depicted in Fig. 1A, this
can result in either an HNF3null allele or
the desired HNF3loxP allele. Southern blot
analysis revealed approximately 20% of the 96 gangcyclovir-resistant
clones tested to be HNF3loxP ES cell clones (data not
shown). Germ line chimeras and mice heterozygous for the
HNF3loxP allele were obtained for two
independent ES cell lines injected. Both lines gave identical results
in the tissue-specific gene ablation experiments.
gene
affects gene function, heterozygous HNF3loxP
mice were bred inter se, and the resulting offspring were genotyped by
PCR analysis. Of 27 offspring genotyped at 4 weeks of age, seven were
HNF3loxP/loxP, in close agreement with the
expected Mendelian frequency (Fig. 1b).
HNF3loxP/loxP mice are fertile, healthy, and
have normal growth curves (data not shown). Thus, insertion of the two
loxP sites into the HNF3 locus does not appear
to interfere with HNF3 function in this genetic background.
Ablation of the HNF3 gene in the liver.
In
order to investigate liver development and function in
hepatocyte-specific HNF3 knockout mice, we utilized a transgenic mouse with Cre under the control of the albumin promoter
(23). HNF3loxP heterozygous mice
carrying this Alb.Cre transgene (HNF3loxP/+;
Alb.Cre) were bred with HNF3loxP homozygous mice,
and embryos were collected from various stages of gestation. As shown
in Fig. 2A through F, no morphological abnormalities were observed in the livers of
HNF3loxP/loxP; Alb.Cre embryos compared to their
wild-type control littermates. Furthermore, adult livers that lack
HNF3 have a normal morphology (compare Fig. 2G and H; also data not
shown). HNF3loxP/loxP; Alb.Cre mice were born in the
expected Mendelian distribution, and no significant differences in body
weights were observed between these mice and their wild-type control
littermates (data not shown). Liver-specific HNF3 knockout mice were
fertile and produced normal-sized litters.
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knockout mice, we
investigated whether we had indeed deleted the HNF3 gene
in the liver. Therefore, steady-state HNF3 mRNA levels were analyzed
in total liver RNA obtained from adult HNF3loxP/loxP;
Alb.Cre or control mice using quantitative RNase protection analysis.
HNF3 message was undetectable in the livers of 10-week-old HNF3loxP/loxP; Alb.Cre mice (Fig.
3A), indicating that the Cre recombinase had efficiently excised the loxP-flanked HNF3 gene in
hepatocytes.
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protein in
livers obtained from HNF3loxP/loxP; Alb.Cre mice and
their control littermates during various developmental stages using
immunohistochemical staining with an antibody specific to HNF3.
Although the endogenous albumin promoter is active during the onset of
liver development (approximately 9.5 days postcoitum [d.p.c.] in the
mouse [4]), HNF3 inactivation was not evident in
livers from midgestation HNF3loxP/loxP; Alb.Cre embryos
(compare Fig. 2A and B and Fig. 2C and D). By 18.5 d.p.c.,
however, the vast majority of hepatocytes from such embryos had lost
expression of HNF3 (compare Fig. 2E and F). HNF3 deletion is
restricted to the liver, as HNF3 is still expressed in other organ
systems where HNF3 is normally expressed (data not shown). Finally,
HNF3 protein is missing in over 99.9% of hepatocytes in 10-week-old
mice (compare Fig. 2G and H), consistent with the absence of HNF3
mRNA shown in Fig. 3A. Thus, we conclude that HNF3 is not required
for the maintenance of hepatocytes in late fetal and postnatal development.
Ablation of HNF3 does not lead to dramatic changes
in the hepatic transcriptional program.
The expression of many
genes required for yolk sac and liver metabolism, including
apolipoproteins, aldolase B, and albumin, was dramatically decreased or
completely absent in embryoid bodies derived from ES cells lacking
HNF3 (12). In addition, expression of HNF3 was absent
and that of HNF1 and HNF4 was reduced dramatically. As visceral
endoderm differentiated in vitro expresses many of the same genes as
hepatocytes in vivo, the notion was put forward that HNF3 directs a
regulatory network of transcription factors and their targets in the
metabolism of yolk sac and liver. However, as shown in Fig.
4A expression levels of mRNAs encoding
apolipoproteins, albumin, and gluconeogenic enzymes are not
significantly reduced in adult livers of HNF3loxP;
Alb.Cre mice, despite the fact that these livers are devoid of HNF3
mRNA and protein.
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also regulates a network of transcription factors in
visceral endoderm differentiated from embryoid bodies in vitro that
includes HNF1, HNF4, and HNF3 (12). Therefore, we
analyzed the steady-state mRNA levels of these genes by quantitative RNase protection assay. Consistent with the findings by Duncan et al.
(12), mRNA levels of HNF3 were unchanged in the livers from mutant animals (Fig. 3A). However, in contrast to the situation in
embryoid bodies, there was no decrease in the transcript levels of
HNF3 or HNF1 but a small (60%) increase in HNF4 relative mRNA
levels in liver (Fig. 3A and B). Furthermore, the relative mRNA levels
for HNF1 and other liver-enriched transcription factors are
unaffected (Fig. 3B and data not shown).
To address the possibility that the remaining HNF3 factors could
compensate for lack of HNF3 function at a posttranscriptional level
via increased protein stabilization or nuclear localization, we assayed
nuclear HNF3 binding activity of hepatocytes that lack HNF3 and
compared these to wild-type controls using an electrophoretic mobility
shift assay with the albumin enhancer eG site, which contains an HNF3
binding site, as a probe. As demonstrated in Fig. 3C, there is no
upregulation of HNF3 or HNF3 nuclear binding activity in
hepatocyte nuclei that lack HNF3 protein.
To identify other potential liver-specific targets of HNF3, we used
mRNA from livers of 10-week-old HNF3loxP/loxP; Alb.Cre
mice and wild-type control littermates and screened for altered
expression profiles using cDNA and expressed sequence tag (EST)
microarray hybridization. As shown in the scatter plot in Fig. 4B, the
overall transcriptional program is strikingly similar in livers
containing or lacking HNF3. Out of 8,700 mouse transcripts analyzed,
three genes were reduced approximately two- to threefold in livers that
lack HNF3 compared to control livers (Fig. 4B), whereas one gene was
induced by more than twofold. To confirm whether differences revealed
by the microarray hybridization were real and not just an artifact of
the assay, the transcript abundance of three of these differentially
expressed genes was analyzed by quantitative reverse transcription-PCR
in livers of HNF3loxP/loxP; Alb.Cre mice and control
mice (Fig. 4C). After first determining the number of PCR cycles
required for exponential amplification for each primer pair (data not
shown), we compared mRNA levels in livers from four wild-type control
and four mutant animals. As shown in Fig. 4C, deiodinase mRNA levels
are induced two-fold in livers that lack HNF3, in close agreement
with the result from the array hybridization. Likewise, -globin mRNA
levels were reduced by approximately twofold, as had been indicated by
the microarray data. These results confirm the usefulness of microarray hybridization for obtaining accurate comparisons of the global transcriptional program between wild-type and mutant animals. These
very limited changes in the overall gene expression pattern between
HNF3 wild-type and mutant livers also argue that HNF3 is not
required for the maintenance of the global hepatic transcriptional program.
Physiological consequence of hepatocyte-specific
HNF3 gene inactivation.
To assess whether loss of
HNF3 in the liver affects liver metabolism of the
HNF3loxP; Alb.Cre mice, we measured various serum
parameters that are indicative of hepatic metabolism. There were no
significant differences in these parameters between
HNF3loxP; Alb.Cre mice and wild-type control
littermates, as summarized in Table 1.
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have decreased or absent expression of the genes
encoding the glycolytic enzymes L-type pyruvate kinase and
aldolase B (12). As HNF3 potentially regulates genes
required for normal glucose homeostasis in vivo, we performed glucose
tolerance tests on adult HNF3loxP; Alb.Cre mice and
wild-type control littermates to examine whether they respond normally
to a glucose challenge. Mice that lack hepatic HNF3 clear excess
blood glucose at a rate similar to that of their wild-type control
littermates (Fig. 5). Therefore, hepatic HNF3 appears not essential for normal glucose homeostasis in vivo.
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) and wild-type control (
)
mice. Blood glucose levels are shown at indicated time points after
intraperitoneal administration of glucose. Values are means ± standard errors of the means (error bars) of seven (control) or nine
(HNF3| |
DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We have employed conditional gene ablation of HNF3
using the Cre-loxP recombination system to generate a mouse model that lacks HNF3 specifically in the liver. While
hepatocyte-specific inactivation of HNF3 was greater than
99.9% efficient in the adult liver, inactivation of HNF3
gene expression was not evident until the end of fetal development,
although the albumin gene is activated on day 9 of gestation
(4). A similar temporal discrepancy between Cre expression
and gene inactivation has been observed when the Pdx1 gene
was ablated in -cells of the pancreas using the Cre-loxP system
(1). Several factors could account for this delay in gene
inactivation. First, plasmid transgene expression levels depend on the
choice of the promoter as well as position effects after insertion of
the transgene into the genome. Secondly, before HNF3 (or PDX1)
protein expression is extinguished, Cre must first be expressed by the
tissue-specific promoter and transported to the nucleus where it must
recombine both loxP-flanked alleles. Finally,
depending on the half-life of the mRNA and protein
remaining after Cre-mediated recombination, hours or days might
be required until expression is completely lost. Therefore, using the HNF3loxP/loxP; Alb.Cre mice
we were unable to determine whether HNF3 has a role in
initiating the hepatic differentiation program during early liver
development, as HNF3 was still present at this time. A
Cre transgene expressed specifically in the prehepatic endoderm may
be used in the future to investigate whether HNF3 is
required for organogenesis of the liver from foregut endoderm.
Analyses of the promoters of liver-specific genes have identified
binding sites for multiple transcription factors, leading to the
hypothesis that cooperation between several liver-enriched transcription factors regulates hepatic gene expression (reviewed in
reference 5). This model would predict that loss of
one transcription factor alone would have minor consequences for the global liver gene expression profile. In contrast to this model, the
idea has been put forth that a transcriptional hierarchy among the
liver-enriched transcription factors exists to maintain the hepatic
transcriptional program (11, 12, 19, 30). Visceral endoderm
expresses many genes encoding metabolic enzymes also active in the
liver. For this reason, the contribution of HNF3 to their regulation
was investigated in visceral endoderm derived from embryoid bodies
lacking HNF3 (12). Under these in vitro conditions, loss of HNF3 resulted in a dramatic
alteration in mRNA abundance of the genes encoding
hepatocyte-enriched transcription factors (HNF3, HNF1, and
HNF4) and their target genes that are involved in normal hepatic
metabolism and differentiation. In contrast, mice lacking
HNF3 in the liver exhibited only minor changes in the
transcriptional program.
Several explanations could account for the mild hepatic phenotype in
HNF3loxP/loxP; Alb.Cre mice. First, compensatory binding
by HNF3 and HNF3 proteins could explain the largely unchanged
hepatic gene expression profile. It has been shown, for instance, that
HNF3 can bind to and transactivate the same
cis-regulatory elements as HNF3 in a native chromosomal
context (12). However, our evidence clearly shows the lack
of upregulation of the remaining HNF3 genes at either the
transcriptional (Fig. 3A) or posttranscriptional level (Fig. 3C) in
livers from HNF3loxP/loxP; Alb.Cre mice, in contrast to
what had been observed in livers from HNF3 mutant animals
(15). Alternatively, hepatic gene expression may be
redundantly regulated by a combination of liver-enriched and ubiquitous
transcription factors, any one of which might play only a minor role in
its regulation. This notion is supported by gene targeting of HNF1,
in which many of the previously known HNF1 targets identified in
vitro were not altered in the mutant animals (24).
In summary, HNF3 gene targeting studies have identified
several steps in mammalian development that are critically dependent on
HNF3. Regarding endoderm development, these include the
formation of a functional node, from which definite endoderm is
derived, the development of foregut and midgut (but not hindgut)
endoderm, and the differentiation of functionally competent visceral
endoderm (2, 10, 33). While the molecular targets of HNF3
in the development of the node and definitive endoderm are largely
unknown, it appears that the activation of HNF4 and HNF1 and
their target genes are dependent on HNF3 in visceral endoderm, at
least in vitro. However, our studies have shown that gene regulation in visceral endoderm and liver are not necessarily parallel, as HNF3 is
not required for the maintenance of the network of HNF transcription factors or for the global transcriptional program in hepatocytes.
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ACKNOWLEDGMENTS |
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N.J.S. and S.-L.A. contributed equally to this work.
We are grateful to M. Birnbaum, L. Greenbaum, and M. A. Lazar for
critical reading of the manuscript; T. Jessell for providing the
HNF3 antibody; K. Zaret, P. Bossard, and L. Greenbaum for providing
assistance with the preparation of nuclear extracts and the EMSA; R. Ahima for assistance with the glucose tolerance test; and G. Schütz for his support during the initial phase of the project.
Our studies were facilitated by the Center for Molecular Studies in Digestive and Liver Disease at the University of Pennsylvania (P30 DK50306). This work was supported by the NIDDK (RO1 DK55342 to KHK and RO1s DK42502 and DK42612 to MAM), the Association pour la Recherche sur le Cancer, the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, and the Centre Hospitalier Universitaire Régional (grant to S.L.A.). N.J.S. was supported through an NIH pre-doctoral training grant (5-T32-GM08216).
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FOOTNOTES |
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* Corresponding author. Mailing address for Klaus H. Kaestner: Department of Genetics, University of Pennsylvania School of Medicine, 415 Curie Blvd., Philadelphia, PA 19104-6145. Phone: (215) 898-8759. Fax: (215) 573-5892. E-mail: kaestner{at}mail.med.upenn.edu. Mailing address for Siew-Lan Ang: Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur, B.P. 163, 67404 Illkirch Cedex, France. Phone: 333 88 653342. Fax: 333 88 653 201. E-mail: siew-lan{at}titus.u-strasbg.fr.
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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, July 2000, p. 5175-5183, Vol. 20, No. 14
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