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First published online May 16, 2007
doi: 10.1242/10.1242/dev.000281
1 Department of Biochemistry and Biophysics, Programs in Developmental Biology,
Genetics and Human Genetics, Liver Center, University of California, San
Francisco, CA 94158, USA.
2 Department of Cell and Developmental Biology, University of Pennsylvania
School of Medicine, Philadelphia, PA 19104, USA.
3 Georges Kohler Laboratory, Max-Planck Institute, 79108 Freiburg,
Germany.
4 Department of Cell Biology, Duke University Medical Center, Durham, NC 27710,
USA.
Author for correspondence (e-mail:
didier_stainier{at}biochem.ucsf.edu)
Accepted 20 March 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: hhex, prox1, alk8 (acvr1), Competence, Endoderm, Hepatocyte, Zebrafish
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Rossi et al., 2001;
Calmont et al., 2006) (reviewed
by Zaret, 2002;
Zhao and Duncan, 2005). An in
vitro mouse embryonic tissue explant system was used to show that Fgfs can
replace the cardiac mesoderm to induce hepatic gene expression
(Jung et al., 1999), that
inhibitors of Fgf signaling block hepatic specification
(Jung et al., 1999;
Calmont et al., 2006), and that
a Bmp inhibitor, noggin, blocks hepatic specification by the STM
(Rossi et al., 2001). The
crucial role of Fgf and Bmp signaling in hepatic specification was also
revealed in chick embryos using chick tissue explant cultures and ex vivo
cell/bead implantation experiments (Zhang
et al., 2004). In tissue explants, FGF2 could substitute for the
cardiac mesoderm to induce hepatic markers such as HHEX and albumin
in the anterior lateral endoderm, and noggin blocked the expression of these
markers (Zhang et al., 2004).
Implantation of noggin-expressing cells into the anterior lateral regions of
chick embryos inhibited HHEX expression in the anterior lateral
endoderm, and that of BMP2-soaked beads induced ectopic HHEX
expression in the endoderm just posterior or lateral to the endogenous
HHEX expression domain (Zhang et
al., 2004).
While Foxa and Gata genes have been shown by genetic analysis to regulate
the competence of foregut endodermal cells to respond to hepatic inductive
signals (Bossard and Zaret,
1998; Cirillo et al.,
2002; Lee et al.,
2005a), no hepatic inducer has been validated genetically. Mice
carrying mutations in individual components of the Fgf or Bmp signaling
pathways either do not survive to the point of hepatic specification
(Yamaguchi et al., 1992;
Deng et al., 1994) or exhibit
no defects in hepatic specification, possibly due to compensation by other
components of the pathway (Weinstein et
al., 1998; Miller et al.,
2000).
In addition to their role in hepatic specification, Fgf and Bmp signaling
regulate the morphogenetic outgrowth of the hepatic endoderm. Data from the
tissue explant system mentioned above indicated that FGF8 and BMP4 also
contribute to the morphogenetic outgrowth of the hepatic endoderm
(Jung et al., 1999;
Rossi et al., 2001), and
analysis of Bmp4 mutant mouse embryos provided genetic evidence for
the role of Bmp4 in liver bud formation
(Rossi et al., 2001).
In zebrafish, hepatoblast specification is thought to occur at
approximately 22 hours post-fertilization (hpf) as marked by the localized
endodermal expression of hhex and prox1
(Ober et al., 2006), two
transcription factor genes also expressed in mouse
(Burke and Oliver, 2002) and
chick (Zhang et al., 2004)
hepatoblasts. Recently, the analysis of prometheus (prt;
wnt2bb - ZFIN), a zebrafish mutant that exhibits profound defects in
hepatoblast specification, was reported
(Ober et al., 2006). The
prt gene encodes a novel Wnt2b homolog, indicating that canonical Wnt
signaling is essential for hepatoblast specification. However, no zebrafish
mutants that implicate Fgf or Bmp signaling in hepatoblast specification have
been reported thus far.
Both Fgf and Bmp signaling play crucial roles in gastrulation and embryonic
patterning (Amaya et al., 1991;
Kishimoto et al., 1997);
therefore, it is essential to block Fgf and Bmp signaling after gastrulation
in order to examine later developmental events such as hepatoblast
specification. In addition, the functional redundancy that stems from the
existence of multiple Fgf and Bmp ligands and receptors may complicate the
identification of mutants defective in hepatoblast specification. Here, we
have overcome these technical and biological challenges by using transgenic
zebrafish lines that overexpress dominant-negative Bmpr1a
(Pyati et al., 2005) or
fgfr1 (Lee et al.,
2005b) under the regulation of the heat shock cognate 70-kd
protein (hsp70) promoter. The dnBMPR and dnFgfr1 proteins can
completely block most, and possibly all, Bmp
(Graff et al., 1994) and Fgf
(Amaya et al., 1991) signaling,
respectively, and hsp70 allows one to temporally control transgene
expression (Halloran et al.,
2000). We show that blocking Bmp or Fgf signaling resulted in
profound defects in hepatoblast specification. Surprisingly, hepatocyte
differentiation occurred in embryos temporarily lacking Bmp signaling,
although with a substantial delay.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We used the following mutant and transgenic lines:
laftm110b (Bauer et
al., 2001; Mintzer et al.,
2001), Tg(hsp70l:dnBmpr-GFP)
(Pyati et al., 2006),
Tg(hsp70l:dnfgfr1-EGFP)
(Lee et al., 2005b),
Tg(lfabp:dsRed) (Her et
al., 2003), Tg(hsp70:alk8) and
Tg(hsp70:bmp2b) fr13
(Chocron et al., 2007).
Heat-shock conditions
Embryos were heat shocked at various stages by transferring them into a
pre-warmed plate containing egg water on a heat block.
Tg(hsp70l:dnBmpr-GFP) embryos were heat shocked for 25
minutes at 40°C; Tg(hsp70l:dnfgfr1-EGFP) embryos for 20
minutes at 37°C. After heat shock, the plate containing the embryos was
transferred into a 28°C incubator, and embryos were harvested between 30
and 40 hpf. As GFP expression is maintained in these embryos for at least 1
day after heat shock, hemizygous embryos expressing GFP were easily sorted.
Heat shock at 40°C (but not 37°C) substantially delays development of
wild-type and hemizygous embryos, making it difficult to estimate the exact
stage of embryos based on incubation time. Thus, we assigned stage based on
gut morphology of wild-type embryos revealed by foxa3 expression
(Field et al., 2003). To
overexpress Bmp2b while blocking Fgf signaling, embryos were heat shocked for
20 minutes at 37°C. Hemizygous
Tg(hsp70:bmp2b)fr13 embryos were easily
distinguished based on the yolk extension defect, and further tested for
presence of the hsp70:bmp2b transgene by PCR using genomic DNA after
in situ hybridization, using the following primers:
5'-CATGTGGACTGCCTATGTTCATC-3' and
5'-GAGAGCGCGGACCACGGCGAC-3'.
In situ hybridization
Whole-mount in situ hybridizations were performed as previously described
(Alexander et al., 1998), using
the following probes: hhex (Ho et
al., 1999), prox1
(Glasgow and Tomarev, 1998),
gata4/5/6 (Reiter et al.,
1999), foxa3
(Odenthal and Nusslein-Volhard,
1998), cp (Korzh et
al., 2001) and pdx1
(Milewski et al., 1998).
Injection of morpholino antisense oligonucleotides
Wild-type embryos were injected at the one- or two-cell stage with 10 ng
gata4 MO and/or 2.5 ng gata6 MO
(Holtzinger and Evans, 2005),
and they were assayed between 37 and 38 hpf.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Wallace and Pack, 2003;
Ober et al., 2006). Their
expression in the zebrafish liver is initiated at approximately 22 hpf and
maintained thereafter (Ober et al.,
2006). To determine whether Bmp signaling is essential for
hepatoblast specification in vivo, we utilized
Tg(hsp70l:dnBmpr-GFP) fish
(Pyati et al., 2005). Embryos
obtained from outcrossing a hemizygous Tg(hsp70l:dnBmpr-GFP)
fish were heat shocked at 18 hpf, a stage before the onset of hhex
and prox1 expression in the liver primordium. We found that the
earliest liver markers, hhex and prox1, were barely
expressed in the liver region of the heat-shocked hemizygous embryos
(Fig. 1B,C,E,F, arrows),
whereas hhex expression in the pancreatic islet and prox1
expression in the interrenal primordium appeared unaffected
(Fig. 1B,C,E,F, arrowheads).
These data indicate that Bmp signaling occurring after 18 hpf is essential for
hepatoblast specification. Hepatocyte differentiation, as assessed by
ceruloplasmin (cp) expression, was also absent in the
heat-shocked hemizygous embryos (Fig.
1Q). In order to visualize the entire endoderm, we examined the
expression of the endodermal marker foxa3
(Odenthal and Nusslein-Volhard,
1998; Field et al.,
2003). Leftward bending of the gut
(Horne-Badovinac et al., 2003)
was often defective in the heat-shocked hemizygous embryos
(Fig. 1N,O; bracket). These
data suggest that Bmp signaling is also required for the morphogenesis of the
gut but not for the maintenance of endodermal gene (e.g. foxa3)
expression. As gata4 and gata6 are expressed in the liver
primordium (see Fig. S1 in the supplementary material), are essential for
hepatocyte differentiation in zebrafish
(Holtzinger and Evans, 2005)
and have been associated with Bmp signaling
(Rossi et al., 2001;
Zaret, 2002;
Zhao and Duncan, 2005), we
examined their expression in the heat-shocked hemizygous embryos. Both
gata4 and gata6 were strongly expressed in the liver region
and weakly expressed in the intestinal endoderm in wild-type embryos
(Fig. 1G,J, arrows and
brackets). In the heat-shocked hemizygous embryos, the expression of
gata4 and gata6 in the liver region was greatly reduced but
not completely abolished, whereas the expression of these genes in the
intestinal endoderm appeared relatively less affected
(Fig. 1H,I,K,L, arrows versus
brackets). These data suggest that Bmp signaling may regulate gata4
and gata6 expression in the liver primordium.
Alk8 is required for early liver development
As our data indicated that Bmp signaling was essential for hepatoblast
specification in zebrafish and a mutation in alk8 (also known as
laf, acvr1 - ZFIN), a type I Bmp receptor, was identified in a
forward genetic screen for genes involved in endodermal organ morphogenesis
(E.A.O., H. Verkade, H. A. Field, P. D. Si Dong, P. Aanstad, T. Sakaguchi, M.
Bagnet, C. Munson, W.-S. Chung, C.H.S., S. Curado, R. Anderson, J. Frantsve,
D. Beis, T. Bartman and D.Y.R.S., unpublished), we next examined liver
development in alk8 mutant embryos
(Bauer et al., 2001;
Mintzer et al., 2001). The
expression of hhex and prox1 appeared greatly reduced in the
mutants at 26 and 34 hpf compared with their wild-type siblings
(Fig. 2A-D, arrows; data not
shown), but the reduction appeared less severe than that seen in embryos in
which Bmp signaling was blocked at 18 hpf
(Fig. 2B,D versus
Fig. 1C,F, arrows), suggesting
that other Bmp receptors or maternally deposited Alk8 might compensate for the
loss of zygotic Alk8 function in hepatoblast specification. The expression of
gata4, gata6 and cp in alk8 mutant embryos appeared
similar to that in embryos in which Bmp signaling was blocked at 18 hpf
(Fig. 2F,H,L), further
suggesting that Alk8 is part of the Bmp signaling machinery regulating
hepatoblast specification. However, the leftward bending of the gut appeared
unaffected in alk8 mutant embryos
(Fig. 2J, bracket), suggesting
that other Bmp receptors or maternally deposited Alk8 function might
compensate for the loss of zygotic Alk8 function in this morphogenetic
process.
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Gata4 and Gata6 are required for the expansion and differentiation of liver progenitors
Gata transcription factors have been reported to play an essential role in
rendering endodermal cells competent to become hepatocytes by unfolding
chromatin (Bossard and Zaret,
1998; Cirillo et al.,
2002). Among Gata transcription factor genes in zebrafish,
gata4 and gata6, but not gata5, are expressed in
the hepatic endoderm (see Fig. S1 in the supplementary material; data not
shown). When both genes were knocked down by morpholino oligonucleotides
(MOs), hepatocyte differentiation, as assessed by transferrin
(transferrin-a - ZFIN) expression at 3 days post-fertilization (dpf),
was completely abolished (Holtzinger and
Evans, 2005). In embryos in which Bmp signaling was blocked at 18
hpf, gata4 and gata6 expression was greatly reduced in the
liver region (Fig. 1H,I,K,L,
arrows). Thus, we hypothesized that the hepatoblast specification defect in
embryos lacking Bmp signaling might result from the reduction of
gata4 and gata6 expression, causing endodermal cells to lose
competence to become hepatoblasts. To test this hypothesis, we injected
gata4 and gata6 MOs, and examined the expression of the
hepatoblast specification markers, hhex and prox1, and a
differentiation marker, cp, at 37-38 hpf. Injections of
gata4 or gata6 MO fully blocked hepatocyte differentiation
in 63 and 71% of the embryos, respectively; injections of both MOs fully
blocked hepatocyte differentiation in 95% of the embryos
(Fig. 5L-N). Injections of
gata4, gata6 or both MOs did not fully block hepatoblast
specification (Fig. 5B-E,G-J),
but the double MO injections significantly reduced hhex expression in
50% and prox1 expression in 69% of the embryos
(Fig. 5E,J). The less severe
defects in the double morphants than in embryos lacking Bmp signaling could be
due to variation in the efficacy of the MO-based depletion. Altogether, these
data suggest that the hepatoblast specification defect in embryos lacking Bmp
signaling may at least partially result from the reduction of gata4
and gata6 expression.
|
).
Embryos obtained from outcrossing a hemizygous
Tg(hsp70l:dnfgfr1-EGFP) fish were heat shocked at 18 hpf, a
stage before the onset of hhex and prox1 expression in the
liver primordium. We found that hhex and prox1 expression in
the liver region was almost absent in the heat-shocked hemizygous embryos,
whereas hhex expression in the pancreatic islet and prox1
expression in the interrenal primordium appeared only weakly affected
(Fig. 6B,C,E, arrows versus
arrowheads), suggesting that Fgf signaling after 18 hpf is essential for
hepatoblast specification. Hepatocyte differentiation, as assessed by
cp expression, was also absent in the heat-shocked hemizygous embryos
(Fig. 6P). The leftward bending
of the gut did not occur in the heat-shocked hemizygous embryos
(Fig. 6M,N; brackets),
suggesting that Fgf signaling is also required for the morphogenesis of the
gut. The expression of gata4 and gata6 in the liver region
was greatly reduced in the heat-shocked hemizygous embryos, whereas their
expression in the intestinal endoderm appeared relatively less affected
(Fig. 6G,H,J,K, arrows versus
brackets), suggesting that Fgf signaling may also regulate gata4 and
gata6 expression in the liver primordium. In general, the liver
defects in embryos in which Fgf signaling was blocked at 18 hpf were similar
to those in embryos in which Bmp signaling was blocked at the same stage.
Fgf signaling does not appear to be essential for the maintenance of specified liver progenitors
We next examined the time-window during which Fgf signaling is required for
hepatoblast specification by blocking Fgf signaling at later stages. When Fgf
signaling was blocked at 22 hpf, hhex and prox1 expression
in the liver region was greatly reduced
(Fig. 7B,C,H, arrows). However,
this expression was slightly higher than that seen in embryos heat shocked at
18 hpf (Fig. 7B,H versus
Fig. 6B,E), and the percentage
of embryos that barely expressed hhex in the liver region was lower
than that in embryos heat shocked at 18 hpf
[Fig. 7C (44%) versus
Fig. 6C (67%)]. In addition,
the expression of gata4, gata6 and cp in the 22 hpf
heat-shocked hemizygous embryos was slightly higher than that in the embryos
heat shocked at 18 hpf (Fig.
7M,N; see Fig. S3B,C,H in the supplementary material;
Table 1). Altogether, these
data suggest that Fgf signaling between 18 and 22 hpf plays an essential role
in hepatoblast specification.
|
), we blocked Fgf signaling at 26 hpf to determine
whether Fgf signaling is required for the maintenance of the specified liver
progenitors. hhex and prox1 were clearly expressed in the
liver region in the hemizygous embryos heat shocked at 26 hpf, although their
expression was reduced compared with wild-type siblings
(Fig. 7D-F,I-K, arrows);
gata4, gata6 and cp expression were also reduced
(Fig. 7P,Q; see Fig. S3E,F,J,K
in the supplementary material; Table
1). However, when Fgf signaling was blocked at 30 hpf,
gata4 and cp expression appeared unaffected (data not
shown). These data suggest that Fgf signaling continues to regulate liver
development after its initial role in hepatoblast specification.
|
|
). DsRed
expression, indicating fabp1a expression, in wild-type siblings was
clearly detected starting at 3 dpf, while it was absent at this stage in
embryos in which Bmp signaling had been blocked at 18 hpf
(Fig. 9I, red circles versus
squares). However, DsRed expression in embryos in which Bmp signaling had been
blocked at 18 hpf was detectable in 33% of the embryos at 5 dpf, and in 90% of
the embryos at 6 dpf (Fig. 9J
and data not shown). Thus, blocking Bmp signaling leads to an apparent lack of
hepatoblast specification, as assessed by hhex and prox1
expression, in about 50% of the embryos at 29-32 hpf
(Fig. 1C,F, arrows). However,
by 6 dpf, 90% of the embryos showed a clear presence of hepatocytes as
assessed by DsRed expression, indicating that endodermal cells maintain
competence to give rise to hepatocytes for longer than anticipated. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Function of Bmp signaling in early liver development
It has been suggested that Bmp signaling plays multiple roles in early
liver development (Rossi et al.,
2001). Initially, Bmp signaling appears to induce or maintain the
expression of Gata4, a competence factor gene, in the mouse foregut
endoderm, suggesting that it regulates hepatic competence. Subsequently, Bmp
signaling appears to induce hepatic specification. Third, Bmp signaling is
required for liver bud formation. The evidence supporting the first and second
roles of Bmp signaling came from in vitro tissue explant experiments, and the
evidence supporting its third role came from the analysis of Bmp4
mutant mouse embryos combined with in vitro tissue explant experiments
(Rossi et al., 2001). In our
study, we provide genetic evidence for the role of Bmp signaling in
hepatoblast specification. By blocking Bmp signaling at 18 and 22 hpf and by
examining alk8 mutant embryos, we reveal the essential role of Bmp
signaling in hepatoblast specification; by blocking at 26 and 30 hpf, after
hepatoblast specification has occurred, we show that Bmp signaling is not
essential for the maintenance of specified liver progenitor cells but
continues to regulate liver development.
|
). FGF1 and
FGF2 appear to be sufficient to induce hepatic specification; FGF8 appears to
regulate the outgrowth of the hepatic endoderm. However, no liver defect has
been described in Fgf1;Fgf2 double mutant mice
(Miller et al., 2000), and
there are no known mouse mutations affecting Fgf signaling that block hepatic
specification or the outgrowth of the hepatic endoderm. Because the mouse
genome appears to encode 22 Fgfs and four Fgf receptor tyrosine kinases
(Itoh and Ornitz, 2004), it is
likely that functional redundancy of these molecules will prevent one from
finding severe liver defects in single or double mutant mice. A recent study
in mouse (Calmont et al., 2006)
showed that overexpressing Spry2, which inhibits signaling from
receptor tyrosine kinases, including Fgf receptors, in nascent hepatic cells
decreases their survival. However, culturing mouse embryos in the presence of
Fgf inhibitors did not appear to affect endodermal cell survival. Here, we
provide in vivo evidence for the role of Fgf signaling in hepatoblast
specification. By blocking Fgf signaling at 18 and 22 hpf, we reveal the
essential role of Fgf signaling in hepatoblast specification; by blocking at
26 and 30 hpf, after hepatoblast specification has occurred, we show that Fgf
signaling is not essential for the maintenance of specified liver progenitors
but continues to regulate liver development.
Source of Bmp and Fgf ligands in zebrafish embryos
While data in mouse support the argument that the Bmp and Fgf signals
involved in hepatic specification are expressed in the STM
(Rossi et al., 2001) and
cardiac mesoderm (Jung et al.,
1999), respectively, data in chick show expression of
BMP2 and several Fgfs (FGF1, 2, 8 and 12) not in
the mesodermal tissues surrounding the hepatogenic endoderm but in the
hepatogenic endoderm itself (Zhang et al.,
2004). Our data do not provide information about the spatial
expression of the inducers; thus, we have to consider Bmp and Fgf genes,
expressed in the endoderm and/or surrounding mesoderm at the time of
hepatoblast specification. Although the expression pattern of all Bmps (at
least seven) and Fgfs (at least 22) has not been investigated, a number of
Bmps (bmp2a, 2b, 5 and 6) and Fgfs (fgf8, 10, 17b
and 24) appear to be expressed in the mesoderm and/or endoderm of the
liver-forming region around the time of hepatoblast specification (ZFIN
expression database). Furthermore, genetic data have suggested that Gdf6a
(Radar), a member of the TGF-ß superfamily, is a ligand of Alk8
(Sidi et al., 2003) and
gdf6a is also expressed in the endoderm at the time of hepatoblast
specification (ZFIN expression database); thus, not only Bmps but other
members of the TGF-ß superfamily have to be considered for their role in
hepatoblast specification.
|
;
Rossi et al., 2001), indicate
that Fgf signaling does not function downstream of Bmp signaling. Our studies
showing that Bmp2b can partially compensate for the loss of Fgf signaling also
suggest that Fgf signaling does not function downstream of Bmp signaling.
However, in order to define the epistatic relationship between these two
signaling pathways, it will be necessary to identify the Bmp and Fgf ligands
required for hepatoblast specification and to analyze their expression in
embryos lacking Fgf and Bmp signaling, respectively.
It will also be important to address whether Wnt signaling functions in
parallel with, downstream of, or upstream of Bmp and Fgf signaling.
Interestingly, another zebrafish homolog of Wnt2b, wnt2ba, which
appears to be essential for pectoral fin development, appears to regulate the
expression of Bmp and Fgf genes (Ng et
al., 2002; Mercader et al.,
2006). Initially, tbx5 expression in the lateral plate
mesoderm appears to be induced by wnt2ba-mediated signaling and
appears to induce the expression of fgf24 and other Fgf genes. Fgf
signaling in turn appears to induce prdm1 expression, which
subsequently appears to induce fgf10 and bmp2b expression in
the lateral plate mesoderm. These data indicate that Wnt signaling is upstream
of both Bmp and Fgf signaling in pectoral fin development
(Ng et al., 2002;
Mercader et al., 2006).
wnt2bb is essential for liver specification
(Ober et al., 2006), and we
tested whether it lies downstream of Bmp or Fgf signaling in this process. We
found that wnt2bb expression in the lateral plate mesoderm in embryos
in which Bmp or Fgf signaling was blocked at 18 hpf appeared unaffected (data
not shown), suggesting that Wnt signaling does not lie downstream of Bmp or
Fgf signaling in hepatoblast specification.
Endodermal cells maintain competence to give rise to hepatocytes in the absence of hepatic inductive signals
In the absence of hepatic inductive signals, endodermal cells have at least
three choices: adopt a different fate such as pancreas, maintain an
uncommitted, competent state, or die. Data from in vitro culture experiments
indicated that endodermal cells adopt a pancreatic fate in the absence of Bmp
(Rossi et al., 2001) or Fgf
signaling (Deutsch et al.,
2001); those from in vivo mosaic analyses suggested that
endodermal cells die in the absence of Fgf signaling
(Calmont et al., 2006).
However, we observed that expression of pdx1, used as a pancreatic
marker in the in vitro culture experiments, was not expanded into the
liver-forming region in embryos lacking Fgf signaling (data not shown) and
that hepatocyte differentiation occurred in alk8 mutant embryos
overexpressing wild-type alk8 at 34 hpf
(Fig. 4R) as well as in embryos
transiently lacking Bmp signaling (Fig.
9). Our data suggest that endodermal cells maintain an
uncommitted, competent state in the absence of inductive signals.
Because there is weak expression of hhex and prox1 in
alk8 mutant embryos (Fig.
2B,D, arrows), it is not clear whether the recovery of hepatocyte
differentiation in those embryos results from the proliferation of a few
specified hepatoblasts or from de novo hepatoblast specification, or a
combination of both. Half the embryos temporarily lacking Bmp signaling showed
a complete absence of hhex and prox1 expression in their
liver-forming region at 30-32 hpf (Fig.
1C,F). Yet hepatocyte differentiation eventually occurred in a
great majority of those embryos, suggesting delayed hepatocyte specification.
Altogether, these data indicate that endodermal cells in the liver-forming
region remain competent to differentiate into hepatocytes, an observation that
may help explain why some wnt2bb mutants eventually form a liver
(Ober et al., 2006).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/11/2041/DC1
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ACKNOWLEDGMENTS |
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|
Footnotes |
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Present address: National Institute for Medical Research, Developmental
Biology, Mill Hill, London NW7 1AA, UK
Present address: Sars Centre for Marine Molecular Biology, University of
Bergen, Norway
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