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First published online 17 July 2008
doi: 10.1242/dev.023010
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Centre National de la Recherche Scientifique, UMR7622 Biologie du Developpement, 9 quai St. Bernard Bât. C, 75005 Paris, France and Université Pierre et Marie Curie, UMR7622 Developmental Biology, 9 quai St. Bernard Bât. C, 75005 Paris, France.
Author for correspondence (e-mail:
silvia.cereghini{at}snv.jussieu.fr)
Accepted 19 June 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Liver specification, vHNF1 (HNF1β, TCF2), Mouse, Zebrafish
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Rossi et al., 2001). The
hepatic endoderm responds to this induction by expressing albumin
(Alb), transthyretin (Ttr) and alpha fetoprotein
(Afp), and generates a nascent hepatoblast population (bipotential
precursors) from which the hepatocytes and biliary cells are derived
(Lemaigre and Zaret, 2004). By
E9, hepatoblasts proliferate to form the primary liver bud. The basement
membrane surrounding the liver bud is then degraded, and hepatoblasts
delaminate from the bud and invade the surrounding STM in a cord-like fashion
(McLin and Zorn, 2006). Aside
from the inductive signals involved in early liver development, little is
known about the transcriptional regulatory circuit within the ventral endoderm
itself that controls hepatic specification.
Gene-targeting studies have implicated several transcription factor genes
in controlling distinct aspects of early hepatogenesis in the endoderm,
including Hex (Hhex - Mouse Genome Informatics),
Gata6 and Prox1. In Hex-deficient embryos, the
hepatic diverticulum is specified but proliferation is reduced and subsequent
migration of hepatoblasts into the STM fails to occur
(Bort et al., 2004;
Martinez Barbera et al.,
2000). Similarly, in Gata6-/- embryos, hepatic
development is disrupted at the liver bud stage soon after hepatic
specification (Zhao et al.,
2005). The homeodomain transcription factor PROX1 acts at later
stages. Hepatoblasts differentiate but are unable to delaminate into the
adjacent STM; this is most likely owing to their inability to degrade the
basement membrane surrounding the hepatic bud
(Sosa-Pineda et al., 2000).
Thus, although mutations in these transcription factors affect early liver
development, in each case defects occur shortly after hepatic induction.
Defective hepatic specification has only been observed in a double-mutant
mouse model in which both Foxa1 and Foxa2 are deleted at the
onset of liver induction (Lee et al.,
2005). Interestingly, ventral endoderm explants of these compound
mutants are unable to induce hepatogenesis in vitro, thus providing genetic
support for the requirement of Foxa factors for hepatic competence. By
contrast, constitutive inactivation of Foxa1 or ablation of
Foxa2 specifically in the ventral endoderm does not affect normal
hepatic specification or differentiation.
Recent studies in mouse and zebrafish indicate that the homeodomain
transcription factor vHNF1 (HNF1β, TCF2) plays a crucial role in the
early development of several endodermally derived organs
(Haumaitre et al., 2005;
Sun and Hopkins, 2001).
vHnf1-deficient mouse embryos die before gastrulation owing to
defective formation of extraembryonic visceral endoderm. This early embryonic
lethality can be rescued by providing vHnf1-null embryos with a
wild-type (WT) extraembryonic endoderm through the use of tetraploid embryo
complementation (Barbacci et al.,
1999; Haumaitre et al.,
2005). Using this technique, we have previously shown that vHNF1
is required for ventral pancreas induction, as well as for normal dorsal
pancreas morphogenesis and regional specification of the gut
(Haumaitre et al., 2005).
Rescued vHnf1-deficient embryos also exhibit a severe liver
hypoplasia, the molecular basis of which remains essentially unexplored.
Interestingly, zebrafish vhnf1 hypomorphic mutants also exhibit
abnormal gut, liver and pancreas development
(Sun and Hopkins, 2001),
suggesting that the function of vHNF1 is conserved through vertebrate
evolution. In this study, we have examined the role of vHnf1 in early
hepatic development in both mouse and zebrafish vHnf1 mutant embryos.
Our results uncover the requirement of vHNF1 for liver specification in both
organisms. More importantly, using an explant culture system, we have also
established that vHnf1-deficient mouse ventral endoderm is unable to
respond to FGF signals, which have previously been shown to be sufficient to
induce hepatic specification in vitro
(Calmont et al., 2006;
Serls et al., 2005).
Therefore, vHnf1 is required for the initiation of liver development
in vertebrates.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The
genotype of 4n embryos was confirmed by PCR analysis of genomic DNA and no WT
allele was detected in embryos derived from vHnf1-/- ES
cells. Control embryos were obtained by parallel matings (OF1 females x
B6D2F1 males). We used embryos with identical numbers of somites to ensure
that they were at equivalent developmental stages.
Immunohistochemistry and in situ hybridization
Antibody staining was performed as previously described
(Haumaitre et al., 2005). We
used the following primary antibodies: mouse anti-E-cadherin at 1:100 (BD
Transduction Laboratories), rabbit anti-laminin at 1:50 (Sigma), goat
anti-HNF4
at 1:150 (C-19, Santa Cruz), rabbit anti-vHNF1 at 1:50 (H-85,
Santa Cruz), rabbit anti-NKX2.1 at 1:100 (H-190, Santa Cruz) and rabbit
anti-phosphohistone H3 at 1:250 (Upstate Biotechnology). Secondary antibodies
were goat anti-mouse IgG Alexa Fluor 546 at 1:500, donkey anti-rabbit IgG
Alexa Fluor 488 at 1:500 (Molecular Probes) and biotinylated goat anti-rabbit
IgG, horse anti-mouse IgG and anti-goat IgG (Vector) followed by
streptavidin-Alexa Fluor 488 at 1:500 (Molecular Probes). TUNEL staining was
performed using the Cell Death Detection Kit (Roche). Whole-mount in situ
hybridization or on sections was performed as previously described
(Haumaitre et al., 2005), with
the exception that embryos were fixed with 60% ethanol/11% formaldehyde and
10% acetic acid. The following cRNA probes were used: vHnf1
(Haumaitre et al., 2005),
Afp, Prox1 (G. Gradwohl, Inserm U682, Strasbourg, France), Foxa2,
Foxa1 and Foxa3 (K. H. Kaestner, Department of Genetics,
University of Pennsylvania School of Medicine, Philadelphia, PA), Shh
(A. P. McMahon, Harvard University, Cambridge, MA), Hex (Bedford et
al., 1993), Gata6 and Gata4 (D. B. Wilson, Washington
University Medical Center, St. Louis, MO); Irx2, Hnf4a, Gata1 and
Hlx probes were generated by PCR.
RNA extraction, RT-PCR, embryo tissue isolation and culture
RNA from dissected ventral endoderm or after its in vitro culture was
extracted using the RNeasy Micro Kit (Qiagen) and subjected to
semi-quantitative RT-PCR as described
(Barbacci et al., 1999). The
volume of each cDNA preparation was adjusted to give similar exponential phase
PCR signal for Gapdh. Primer sequences are available upon request.
For explant culture experiments, ventral endoderm was dissected from 4- to
6-somite embryos and cultured for 48 hours at 37°C in 4 microwells (Nunc)
coated with type I collagen (Collaborative Biomedical Products) in 5%
CO2 in DMEM medium supplemented with 10% calf serum, human
recombinant bFGF (Invitrogen; 5 ng/ml) and heparan sulfate proteoglycan
(Sigma; 50 ng/ml).
Zebrafish line maintenance, genotyping and whole-mount in situ hybridization
Zebrafish (Danio rerio) were raised and genotyped as described
(Lecaudey et al., 2007).
Whole-mount in situ hybridization was performed using the following probes:
hhex and transferrin (M. Pack, The Children's Hospital of
Philadelphia, Pennsylvania, PA), gata 4 and gata6 (T. Evans, Albert
Einstein College of Medicine, New York, NY), foxa3, ceruloplasmin and
prox1 (Y. R. Stainier, University of California, San Fransisco, CA)
axial (foxa2) and shh (F. M. Rosa, Inserm U784,
Paris, France).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Thus,
vHnf1 expression demarcates the early hepatic and ventral pancreas
endoderm, which is consistent with a function of this factor during the
earliest stages of development.
Lack of vHNF1 leads to a severe defect in mouse liver development
We have rescued the early embryonic lethality of vHnf1 homozygous
mutants by providing WT extraembryonic endoderm through tetraploid embryo
complementation. In these experiments, the embryo proper is derived
exclusively from vHnf1-/- diploid ES cells and the
visceral endoderm derives from WT tetraploid embryos
(Barbacci et al., 1999).
vHnf1-/- ES cell-derived embryos (henceforth denoted
vHnf1-/-) exhibited apparently normal external
characteristics up to E13.5, except for severe liver hypoplasia, evident by
external inspection (Fig. 2A).
Dissection of mutant livers indicated a 70% reduction in size; yet they
presented the same number of lobes as WT livers
(Fig. 2B).
vHnf1-/- embryos died around E14.5-15.5, probably because
of the inability of the hypoplastic mutant liver to conduct its embryonic
hematopoietic function.
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Analysis of the percentage of cells positive for the mitotic marker phosphohistone H3 (P-H3) showed a 60% decrease in P-H3-positive cells in vHnf1-/- relative to WT livers between E10.5 and E12.5, and a decrease of 60-70% by E13.5 (not shown). Whereas no increase in apoptosis was detected in mutant livers from E10.5 to E12.5, massive apoptosis was detected at E13.5 (Fig. 2H).
Considering that vHnf1 is selectively expressed in the gut endoderm and hepatoblasts, but not in the STM, we examined the expression of early endoderm differentiation markers, including Alb, Afp, Ttr, Prox1, Foxa1, Foxa2, Foxa3, Hnf1a, Hnf4a and Hnf6 (Onecut1 - Mouse Genome Informatics), at earlier stages, before liver damage was evident. The combined expression of these genes would reflect correctly differentiated hepatoblasts. Surprisingly, none of these markers was expressed at E10.5, nor at any stage (Fig. 3A-L, and data not shown), in the vHnf1 mutant liver.
We then examined the expression of markers of other cells types that
compose the embryonic liver, namely the STM, endothelial and hematopoietic
cells. In the embryonic liver, the homeobox transcription factor Hlx
is expressed exclusively in cells derived from the STM
(Hentsch et al., 1996). Once
hepatoblasts invade the STM, Hlx expression essentially becomes
restricted to the periphery of hepatic lobes (E10.5-11;
Fig. 3M). Surprisingly,
Hlx expression at E10.5 was observed homogenously throughout the
entire lobes of vHnf1-/- livers
(Fig. 3N), along with reduced
expression of Gata1 (Fig.
3P), a transcription factor restricted to hematopoietic cells
(Palis et al., 1999).
Together, these results show that mutant hepatic lobes are devoid of
hepatoblasts and are mainly composed of mesenchymal and hematopoietic cells,
which enter into apoptosis by E13.5. These observations further suggest a
defect in hepatoblast formation and/or migration in
vHnf1-/- embryos.
vHNF1 is necessary for hepatic bud formation
Whole-mount in situ hybridization at E9.5 (20-22s) showed that
Afp, one of the first markers expressed at high levels in the hepatic
bud (Fig. 4A), was absent from
the vHnf1-/- mutant presumptive hepatic domain
(Fig. 4B). Foxa2, a
gene normally expressed in the foregut-midgut endoderm and necessary for its
development (Dufort et al.,
1998), was specifically absent from the presumptive hepatic
endodermal region, while in the gut endoderm its expression was caudally
expanded (Fig. 4C,D). In the
vHnf1-/- mutants, the expression of Shh, a
foregut marker, was expanded and higher in the foregut region compared with WT
embryos (Fig. 4E,F), which was
more obvious on sagittal sections (not shown).
In situ hybridization of transversal sections at E9.5 (20-22s) showed that
the expression of Hex, one of the earliest factors expressed in
prehepatic endoderm and required for hepatic and ventral pancreatic bud
development (Bort et al., 2004;
Martinez Barbera et al.,
2000), was barely detectable in the ventral endoderm of
vHnf1-/- embryos (Fig.
4G,H). Yet, Hex expression was not affected in the
thyroid domain (data not shown). At this stage, migration of hepatoblasts is
manifested by progressive disruption of the laminin-positive basal membrane
that surrounds the liver bud of WT embryos
(Fig. 4I). As in Hex
mutants, in vHnf1-/- embryos the formation of a
pseudostratified epithelial hepatic bud was not observed. The ventral endoderm
remained columnar as the epithelium lining the gut tube and was surrounded by
an intact laminin basal membrane (Fig.
4J). In agreement with the lack of endoderm outgrowth and
expansion, we observed a 40% decrease in the number of P-H3+ cells
in the presumptive vHnf1-/- hepatic endoderm
(4.4±1.29%; 17 sections, n=2 embryos) as opposed to WT
controls (7.49±1.35%; 16 sections, n=2 embryos) (data not
shown). At E9.5, Shh is normally expressed in the lateral gut
endoderm in a ventral-dorsal gradient but is excluded from the hepatic bud
(Fig. 4K). In
Hex-/- embryos, ectopic expression of Shh in the
hepatic bud has been proposed to impair the transition of the endoderm to a
pseudostratified epithelium (Bort et al.,
2006). Shh was expressed ectopically and at higher levels
in vHnf1-/- than WT embryos in the lateral gut epithelium
(Fig. 4, compare E,K with F,L);
it was however excluded from the presumptive hepatic bud domain.
|
), but from E9 (14s) is rapidly downregulated in
the prehepatic endoderm while persisting in the gut duodenal region
(Bort et al., 2006) (see also
Fig. S1 in the supplementary material). Surprisingly, Gata4, which
was, as in the WT, induced at 8s and downregulated by 14s in mutant ventral
endoderm (see Fig. S1 in the supplementary material), was subsequently
ectopically expressed in the entire ventral gut of
vHnf1-/- embryos (Fig.
4P and see S1 in the supplementary material). Interestingly, a few
cells coexpressing GATA4 and the pancreatic-duodenal factor PDX1 were observed
in a restricted part of the mutant ventral gut (see Fig. S1 in the
supplementary material).
Remarkably, the expression of Foxa1 and Foxa2 was
strongly decreased and/or absent in the ventral part of the gut and in the
presumptive liver bud of vHnf1-/- embryos
(Fig. 4T,V), whereas their
expression in the lateral gut epithelium was unaffected. Furthermore, the
expression of Foxa3 was completely lost
(Fig. 4X), which is in
agreement with previous data indicating that vHNF1 is a direct transcriptional
activator of this gene in visceral endoderm formation
(Barbacci et al., 1999;
Hiemisch et al., 1997).
These results indicate that vHNF1 is required in two steps of early liver development: the thickening of the hepatic bud and the expression of essentially all known hepatic genes.
vHNF1 is required for hepatic specification of the ventral endoderm
The observation that the expression of Foxa factors is strongly reduced or
absent in the presumptive hepatic endoderm of vHnf1-/-
embryos suggested a defective hepatic specification. To directly address
whether the specification process was impaired, we determined the expression
levels of several hepatic genes in ventral endoderm at the 6-8s stage by
semi-quantitative RT-PCR (Fig.
5A). At this stage, the ventral foregut endoderm is instructed by
FGF and BMP signals to be specified to the hepatic lineage. As expected,
vHnf1 transcripts were detected in ventral endoderm of control
embryos, but not in vHnf1-/- ventral endoderm. Comparable
Hex and Foxa2 transcript levels were detected in mutant and
WT embryos. By contrast, no transcripts of Alb were detected in
mutant ventral endoderm (Fig.
5A). Since the Alb gene encodes the earliest marker
expressed in the prehepatic endoderm, our results demonstrate that vHNF1 is
required for hepatic specification. The same results were obtained when these
analyses were performed at the 8-10s (Fig.
5B) or 10-12s stage (not shown), thereby excluding the possibility
of a delay in hepatic specification. Furthermore, transcripts of
Ptf1a, a key transcription factor required for the acquisition of a
pancreatic fate (Kawaguchi et al.,
2002), were not detected in mutant ventral endoderm at the 8-10s
stage (Fig. 5B). These results
show that ventral pancreas specification does not occur in
vHnf1-/- mutants. This is in agreement with the reported
absence in vHnf1-/- mutants of a ventral pancreas bud at
all stages examined (Haumaitre et al.,
2005), and further indicates a global defect in ventral endoderm
specification.
|
). We
therefore compared the expression of vHNF1 and HNF4 proteins, which at
8s are both strongly expressed in the extraembryonic visceral endoderm. We
found, however, a similar positioning of this tissue relative to the ventral
foregut endoderm and cardiac mesoderm in vHnf1-/- and WT
embryos (Fig. 5, compare C with
D and E with F).
Another possible explanation for defective hepatic specification is a
switch in the identity of the ventral endoderm to another, more-anterior fate
of the foregut, as might be expected from the posterior expansion of
Foxa2 (Fig. 4D) and
Shh (Haumaitre et al.,
2005) expression in the gut of vHnf1-/-
embryos. We analyzed at 8s the expression of several factors typically
expressed in different ventral foregut regions, including Nkx2.1,
which is expressed in the thyroid and lung domain
(Deutsch et al., 2001),
Hex, which is expressed in the thyroid and hepatic endoderm domain
(Martinez Barbera et al.,
2000), and Irx2, which is expressed in the lungs and
tracheapharyngeal domain (Becker et al.,
2001). No differences in the expression pattern of these markers
were observed between mutant and WT embryos, indicating that at this stage,
vHnf1-/- ventral endoderm is apparently correctly
regionalized (Fig. 5G-L). We
also observed correct expression of Hex in the presumptive hepatic
domain of mutant ventral endoderm. However, and in contrast to the similar
levels of Foxa1/2 transcripts observed by RT-PCR analysis of control
and mutant ventral endoderm, the expression of both Foxa1 and
Foxa2 appeared significantly reduced specifically in the mutant
ventral endoderm region (Fig.
5N,P). One possible explanation for this discrepancy is that
during ventral endoderm microdissection, some foregut-adjacent tissue,
expressing normal levels of Foxa1/2 genes, was inadvertently included
and therefore biased our quantifications. More importantly, a further decrease
in Foxa1/2 expression was observed in 14s stage mutant ventral
endoderm (see Fig. S2 in the supplementary material). Moreover, the induction
of Foxa3 expression was severely perturbed as indicated by rare
Foxa3-positive cells distributed throughout the
vHnf1-/- ventral endoderm
(Fig. 5R). These data together
clearly indicate a requirement of vHNF1 for maintaining the expression of
Foxa1 and Foxa2 specifically in the ventral endoderm, as
well as for Foxa3 induction.
|
;
Deutsch et al., 2001;
Lee et al., 2005). Our
experiments show that, in contrast to WT embryos, vHnf1-/-
ventral endoderm (n=3 embryos) was not competent to induce the
expression of Alb or Ttr
(Fig. 5S). These data suggest
that the failure of hepatic specification in the vHnf1-/-
embryo is, at least in part, owing to a loss of competence to respond to FGF
inductive signaling.
|
). Between 24 and 28 hpf, two thickened regions emerge from
the intestinal rod, the anterior of which corresponds to the liver bud and the
posterior to the pancreas bud (Field et
al., 2003). Recent studies point to a conserved molecular program
orchestrating liver formation in this organism. As in amniotes, FGF and BMP
signaling pathways have been shown to be required for liver induction
(Shin et al., 2007). Moreover,
a role for Wnt signaling from the lateral plate mesoderm in zebrafish liver
induction has been recently described
(Ober et al., 2006).
Additionally, in contrast to in amniotes, Hedgehog signaling appears to
regulate the proliferation of already specified liver progenitors
(Wallace and Pack, 2003). It
has also been reported that hhex is required for liver development
and normal gut looping in zebrafish
(Wallace et al., 2001),
whereas gata4 and gata6 play an important, yet redundant,
role in liver expansion and differentiation
(Holtzinger and Evans,
2005).
Several vhnf1 zebrafish mutants presenting abnormal liver and
pancreas development, in addition to defective hindbrain segmentation, have
been isolated (Song et al.,
2007; Sun and Hopkins,
2001). These mutants present a wide range of hepatic phenotypes
from reduced liver and abnormal biliary development
(Matthews et al., 2004;
Sun and Hopkins, 2001) to the
lack of a discernible liver at 3 dpf (Song
et al., 2007). These observations prompted to us to examine in
this organism the role of vhnf1 at the stage of hepatic
specification. For this analysis we chose the
vhnf1hi2169 mutants because they are highly
hypomorphic leading to null alleles (Bagnat
et al., 2007; Sun and Hopkins,
2001).
Although the general expression pattern of vhnf1 in zebrafish
embryos has been reported previously (Song
et al., 2007; Sun and Hopkins,
2001), its early expression in the endoderm before and during
hepatic budding is less well documented. As shown in
Fig. 6A,B, at both 18s and
22-24s stages, vhnf1 was strongly expressed in the pronephric duct,
while weakly expressed in the gut endoderm. By 48 hpf, vhnf1 was
expressed in the gut and in the liver and pancreas anlagen
(Fig. 6S), displaying an
endoderm expression pattern similar to that of foxa3
(Fig. 6O)
(Field et al., 2003).
|
), indicating that vhnf1 is also required for gut
morphogenesis. Together, these data strongly suggest that
vhnf1hi2169 mutant embryos exhibit defective hepatic
specification, whereas the gut endoderm integrity appears to be
maintained. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Unlike Prox1 mutants, in E13.5 vHnf1-/- embryos
the liver architecture is severely disrupted and followed by massive
apoptosis. Since vHnf1 is not expressed in the STM, these
observations imply that signals from hepatoblasts play a crucial role in the
maintenance and generation of the hepatic architecture required for normal
liver function.
Analysis at the time of liver induction indicates that vHNF1 is required
for the acquisition of competence of the ventral endoderm to respond to
inductive signals and for hepatic specification. Remarkably, the liver
induction defect exhibited by vHnf1-/- embryos is
basically identical to that reported in compound Foxa1/Foxa2 mutants.
In both cases, the ventral endoderm fails to acquire a hepatic fate when
cultured in vitro under conditions reported to be sufficient to induce its
specification. The acquisition of hepatic competence is disrupted even though
Foxa1 and Foxa2 transcripts are present, albeit at lower
levels, in vHnf1-/- ventral endoderm. Thus, vHNF1, like
Foxa factors, mediates regional competence of the ventral endoderm to respond
to FGF signaling. Because of their ability to open highly condensed chromatin
structure upon binding, it has been proposed that FOXA and GATA factors behave
as `pioneer factors' that mark their target genes as competent to be expressed
when exposed to the appropriate inductive signals
(Cirillo et al., 2002). Given
the structural properties of the protein it is unlikely that vHNF1 mediates
competence by relieving chromatin condensation. One explanation that we
explored was whether vHNF1 is a direct target of the FGF signaling pathway.
Prior studies in zebrafish supported this hypothesis, as injected
vhnf1 mRNA can rescue the abnormal pancreatic and liver phenotype
caused by the disruption of either FGF or BMP signaling pathways
(Song et al., 2007),
indicating that vhnf1 is downstream of these pathways. Moreover,
recent studies in mice have shown that hepatic gene induction is elicited by
an FGF/MAPK pathway, whereas the FGF/PI3K pathway is required for growth and
morphogenesis of the hepatic bud (Calmont
et al., 2006). In vHnf1-/- mouse embryos, both
processes are affected as hepatic specification is disrupted and the liver bud
fails to form. However, when ventral endoderm was cultured under different
conditions, we found that vHnf1 expression is influenced neither by
FGF signaling nor by pharmacological inhibitors of FGF signaling, indicating
that vHNF1 is not a direct target of FGF signals. Moreover, Fgfr2,
sprouty 2, Dusp6 (Mkp3) and Socs3 genes were
induced at similar levels in vHnf1-/- and WT ventral
endoderm (see Fig. S4 in the supplementary material), suggesting that the
hepatic competence defect of our mutants is not due to a global disruption of
FGF signaling. Based on these observations, we propose that vHNF1 could
mediate competence of the endoderm to respond to FGF signaling by controlling
the expression of a downstream target of this pathway. An alternative,
non-mutually exclusive possibility is that the reduced expression of
Foxa1 and Foxa2, along with the absence of Foxa3,
lead to a level of Foxa proteins that is below the threshold required for
relieving chromatin condensation and subsequent acquisition of the hepatic
competence. Considering the broad expression of both vHnf1 and
Foxa1/2 genes within the gut endoderm, it is difficult to explain how
these factors, acting either in synergy or in a linear cascade, could mediate
hepatic specification in a restricted domain of the ventral endoderm. It
remains possible that local signals induce a hepatic specification factor(s)
or co-factor(s) that either cooperates with vHNF1 or is an obligatory partner
of this gene.
Following the competence step, vHNF1 appears to function through sequential
and complementary mechanisms. One of them concerns the proper regional
specification of the gut and the subsequent accurate acquisition of cell
fates. Abnormal regional gut specification in vHnf1-deficient mutants
at E9.0 is manifested by the caudal expansion of Foxa2 and
Shh expression, along with the ectopic expression of Gata4
in the presumptive hepatic domain, which otherwise essentially lacks all early
hepatic markers examined. One interesting possibility is that ectopic
expression of Gata4 reflects a change in the identity of the ventral
endoderm into a duodenal-like fate. In agreement with the acquisition of a
duodenal fate is the observation of sparse cells coexpressing PDX1 and GATA4
in the presumptive hepatic endoderm at E9. Since a ventral pancreas bud is not
formed at any stage in vHnf1 mutant embryos
(Haumaitre et al., 2005),
these PDX1-positive cells might represent duodenal precursors. A similar
conversion of ventral pancreas progenitor cells into duodenal cells has been
observed in mouse Ptf1a mutants
(Kawaguchi et al., 2002).
Interestingly, Ptf1a is not induced in vHnf1-/-
ventral endoderm at any developmental stage
(Fig. 5)
(Haumaitre et al., 2005). Such
cell-fate conversion would account, at least in part, for the abrogated
morphogenesis and the lack of liver bud outgrowth.
Unlike the strongly downregulated expression of early hepatic genes at
E9.0, at the 8-10s stage, Foxa1, Foxa2 and Hex are
expressed, albeit at different levels, in the presumptive hepatic domain of
vHnf1 mutants. Thus, vHNF1 is not required for the initial induction
of these genes but rather for maintenance of their expression. These
observations further suggest that vHNF1 and FOXA1 and FOXA2 factors do not act
in a simple linear cascade that leads to the activation of liver-specific
genes, but rather in synergistic pathways
(Fig. 7) to activate these
genes. This phase of induction of Foxa genes is shortly followed by a
maintenance phase in which vHNF1 is required both for their sustained
expression and for subsequent induction of other hepatic transcription
factors, thus establishing a complex cross-regulatory network involved in the
determination of the hepatic fate
(Cereghini, 1996). Consistent
with this model (Fig. 7),
functional HNF1 (HNF1A - Mouse Genome Informatics) binding sites in the
regulatory sequences of both Foxa2
(Kyrmizi et al., 2006) and
Foxa3 (Hiemisch et al.,
1997) have been identified. In addition, in silico analysis has
indicated the presence of potential HNF1 binding sites within the upstream
sequences of the Foxa1 gene.
More importantly, a comprehensive analysis of promoter occupancy during
mouse hepatic development from E14 has recently shown that vHNF1, but not the
structurally related protein HNF1, is recruited to the promoter regions of a
`core' group of transcription factors including FOXA2, HNF1, HNF4 (HNF4A),
HNF6 and GATA6, which compose autoregulatory and cross-regulatory circuits
(Kyrmizi et al., 2006).
Interestingly, the complexity and stability of this network increase gradually
during organogenesis (Kyrmizi et al.,
2006).
As in mice, in zebrafish vhnf1 mutant embryos neither the liver
nor the pancreas bud is formed and the gut endoderm is abnormally regionalized
(see Fig. S3 in the supplementary material)
(Sun and Hopkins, 2001).
Despite the fact that in these two organisms the positioning of the gut
endoderm relative to the adjacent tissues and the inductive signals are not
the same (Ober et al., 2006),
our analyses in zebrafish confirm an evolutionary conserved role of
vHnf1 in hepatic specification and development. Interestingly, the
later roles of vHNF1 in intrahepatic biliary morphogenesis are also conserved
in zebrafish (Matthews et al.,
2004). Therefore, vHNF1 represents the earliest transcription
factor involved in zebrafish hepatic specification.
Together, these studies support the notion that vHNF1 acts at two levels
during vertebrate organogenesis. First, in the early acquisition of a hepatic
and pancreatic fate from the multipotent ventral endoderm. Second, in the
normal epithelial morphogenesis of tubular structures [i.e. biliary duct
(Coffinier et al., 2002),
stomach epithelium (Haumaitre et al.,
2005) and gut lumen in zebrafish
(Bagnat et al., 2007)].
Interestingly, these dual functions of vHNF1 correlate with its dynamic
embryonic expression pattern, being initially high throughout the entire liver
and pancreas buds and subsequently restricted to the branched pancreatic
ductal network and the biliary system.
The strategies used to differentiate embryonic stem cells into endodermal
cells are based on the knowledge of the conserved molecular network that
controls endoderm formation in different vertebrate embryos
(D'Amour et al., 2005). From a
therapeutic point of view, it is important to now precisely define how the
endoderm is patterned and how particular organs are induced. Our studies
provide insights into the early molecular events of liver specification in
vertebrates and may contribute to the development of in vitro strategies for
the generation of hepatic cells for regenerative medicine, either from
embryonic stem (Gouon-Evans et al.,
2006) or somatic stem cells.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/16/2777/DC1
|
ACKNOWLEDGMENTS |
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|
Footnotes |
|---|
Present address: Unitat de Biologia Cellular I Molecular, Institut
Municipal d'Investigacio Medica, Barcelona, Spain
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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