1 Department of Molecular and Human Genetics, Baylor College of Medicine, One
Baylor Plaza, Houston, TX 77030, USA
2 Department of Molecular and Cellular Biology, Baylor College of Medicine, One
Baylor Plaza, Houston, TX 77030, USA
3 Program in Developmental Biology, Baylor College of Medicine, One Baylor
Plaza, Houston, TX 77030, USA
* Author for correspondence (e-mail: jamrich{at}bcm.tmc.edu)
Accepted 14 April 2004
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SUMMARY |
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Key words: BMP4, FoxF1, Forkhead, Gut, Lateral plate mesoderm, Morpholino, Visceral mesoderm
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Introduction |
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Xenopus FoxF1 (El-Hodiri et
al., 2001; Koster et al.,
1999
) is the ortholog of the human FREAC-1
(Clevidence et al., 1993
;
Hellqvist et al., 1996
;
Larsson et al., 1995
), the
murine and chick HFH-8 (Funayama
et al., 1999
; Peterson et al.,
1997
) and the Drosophila biniou
(Zaffran et al., 2001
).
Because of relatively recent genome duplication in X. laevis, most
Xenopus genes are present in two copies per haploid genome. These two
copies are usually labeled as `a' and `b'. Typically, the `a' and `b' genes
have the same expression pattern and function. Since previously published
studies show that FoxF1a and FoxF1b have the same expression
pattern (El-Hodiri et al.,
2001
; Koster et al.,
1999
), we will treat them as the same. However, for the sake of
accuracy, we should mention that these studies were performed using
FoxF1b.
Xenopus FoxF1 is first activated during gastrulation in the
presumptive ventro-lateral mesoderm. During neurulation, FoxF1
expression becomes restricted to the lateral plate mesoderm
(El-Hodiri et al., 2001;
Koster et al., 1999
). This
expression is very similar to the expression of its murine ortholog
HFH-8 (Foxf1) (Peterson
et al., 1997
) with some species-specific differences. While both
of the genes are expressed in the lateral plate mesoderm and later in the
visceral mesoderm, the murine Foxf1 is intensely transcribed in the
extra-embryonic mesoderm of allantois, amnion and yolk sack, structures that
do not exist in Xenopus embryos. Interestingly, even the
Drosophila gene biniou displays a highly conserved
expression in the visceral mesoderm. The unique expression pattern of FoxF
genes in the lateral plate/visceral mesoderm makes them important targets for
evaluation of function in order to shed light on their role in gut
development. In vertebrates, the function of Foxf1 has been
investigated only in mice. A targeted elimination of Foxf1 resulted
in defects in mesodermal differentiation and incomplete separation of
splanchnic and somatic mesoderm (Mahlapuu
et al., 2001b
). Analysis of Foxf1 function in older mouse
embryos was hampered by the fact that the Foxf1 deficient embryos
have severe defects in extra-embryonic structures, and these animals die of
apoxia by embryonic day (E) 10.
In invertebrates, the function of FoxF has been investigated in
Drosophila. The Drosophila FoxF gene biniou
(bin) is activated in the trunk visceral mesoderm primordia and has a
role during specification and differentiation of the trunk visceral mesoderm.
Its function is essential for the differentiation of the splanchnic mesoderm
into midgut musculature (Zaffran et al.,
2001), suggesting that the FoxF gene family might have an
important, evolutionarily conserved, function in the development of the
lateral plate/visceral mesoderm.
To gain a better understanding of the function of FoxF genes in
vertebrates, we have decided to monitor the effects of FoxF1 absence
on gut development in X. laevis. We argued that in this developmental
system the interactions with the mother's circulatory system are not required
and therefore the analysis of FoxF1 function is not going to be
hampered by early deaths of embryos. Xenopus embryos are a very good
system for studying gut development, as these embryos are easily accessible at
all developmental stages and the gut development has been described in precise
detail (Chalmers and Slack,
1998,
2000
). In our study, we used a
morpholino oligonucleotide-based approach to interfere with FoxF1
function. Morpholinos are modified oligonucleotides that can inhibit
translation of the target mRNA (Heasman et
al., 2000
). They are resistant to degradation and therefore are
present in embryos for several days after injection. Since injections of
morpholinos provide substantial reduction of the targeted gene product, this
type of interference is often called a gene `knockdown'. Using this approach,
we found that FoxF1 is critical for the proliferation and
differentiation of the lateral plate/visceral mesoderm.
Xenopus FoxF1 is a target of BMP4 signaling and its function is
required for normal activation of Xbap and -smooth muscle
actin. The cis-regulatory elements of FoxF1 required for
BMP4-mediated activation are located within 2 kb, upstream of the 5'
coding region. These sequences are able to direct expression of a GFP reporter
cassette into the lateral plate mesoderm of transgenic tadpoles. For the first
time, the identification of these elements provides a tool to specifically
manipulate gene expression only in the mesodermal component of the gut.
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Materials and methods |
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Constructs for transgenesis
A 2 kb EcoRI-EcoRI DNA fragment upstream of the
transcription start site of FoxF1 was screened out from the
Xenopus genomic library. For the GFP reporter construct, this
fragment was subcloned into pBS-GFP (Zhang
et al., 2003) to generate pBS-FoxF1-GFP. For the lacZ
reporter construct, a BamHI-NotI fragment containing nuclear
localization sequence (NLS) and ß-galactosidase (lacZ) from
pCS2+nßgal was subcloned into pBluescriptIISK and then the 2 kb
EcoRI-EcoRI FoxF1 5' fragment was subcloned
into the EcoRI site to generate pBS-FoxF1-NLS-ßgal. DNA for
transgenesis was prepared by digestion with SacII-PvuI for
pBS-FoxF1-GFP and NotI-HindIII for
pBS-FoxF1-NLS-ßgal.
Western blotting
For in-vitro transcription/translation of FoxF1, we used the TNT
Coupled Reticulocyte Lysate System (Promega) according to the manufacturer's
instructions. SDS-PAGE was performed using a 10% polyacrylamide gel. The
proteins were transferred to a nitrocellulose membrane (Schleicher &
Schuell), and detected with the monoclonal mouse anti-myc antibody 9E10
(Invitrogen) using the ECL Western Blotting Detection Reagents and Analysis
System (Amersham Biosciences).
In-situ hybridization, immunostaining, ß-galactosidase staining and histology
Whole-mount in-situ hybridization was performed as described by Harland
(Harland, 1991).
Digoxigenin-labeled probes were generated from the following plasmids: a
FoxF1 clone containing 99 base pairs of 5' UTR and the complete
coding sequence in the pCRII-TOPO vector was linearized with XbaI and
transcribed with SP6 RNA polymerase; Xbap
(Newman and Krieg, 1998
).
Immunostaining of paraffin sections and whole embryos was performed as
previously described (El-Hodiri et al.,
1997
). Primary and secondary antibodies were used at the following
dilutions: 12/101, 1:200 (Developmental Studies Hybridoma Bank);
-smooth muscle actin, 1:400 (Sigma); HRP-conjugated sheep anti-mouse
IgG, 1:200 (Sigma). Embryos were dehydrated in ethanol, embedded in paraffin
wax and 12 µm sections were cut. Sections were de-waxed in xylene and
mounted with Permount (Fisher). Nuclei were stained with 2 µg/ml Hoechst
33342 (Sigma). ß-galactosidase activity was detected as described by
Turner and Weintraub
(1994
).
TUNEL and BrdU incorporation assays
Apoptotic cells were identified by whole-mount TUNEL staining following a
protocol by Zhang et al. (Zhang et al.,
2003). BrdU incorporation was performed as previously described
(Hardcastle et al., 2000
) with
some modifications on the injection sites: 10 nl of 5-bromo-deoxyuridine
(BrdU) (Roche) were injected into both sides of the neural plate and lateral
plate of stage 19 embryos.
RNA isolation and RT-PCR assay
Preparation of total RNA from animal caps using TRIzol reagent (Invitrogen)
was carried out according to the manufacturer's instructions. RT-PCR was
performed by using the following primers and cycling condition: FoxF1
(55°C, 30 cycles; forward, 5'-AACCCTCTGTCCTCCAGCCT; reverse,
5'-GGTTAGTGCAATGACTAACTTC), Xbra (55°C, 30 cycles; U:
5'-GGATCGTTATCACCTCTG; D: 5'-GTGTAGTCT GTAGCAGCA), EF-1
(55°C, 26 cycles U: 5'-CAGATTGGTGCTGGATATGC; R:
5'-ACTGCCTTGATGACTCCTAG).
Transgenesis
Transgenic Xenopus laevis embryos were generated by restriction
enzyme-mediated integration (REMI) as described
(Amaya and Kroll, 1999;
Kroll and Amaya, 1996
) with
some modification (Zhang et al.,
2003
).
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Results |
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FoxF1 is required for normal proliferation of the lateral plate mesoderm
While the abnormal degree of differentiation alone could explain the lack
of cohesiveness of the smooth muscle layer, a reduced cell proliferation or
increase in cell death in the developing lateral plate mesoderm could be other
contributing factors. Alteration in either of these processes could lead to a
reduced number of mesodermal cells during smooth muscle formation. To evaluate
whether cell proliferation was affected in FoxF1 knockdown
experiments, we used BrdU incorporation to visualize proliferating cells in
these embryos. While cell proliferation was high in the lateral plate mesoderm
of control morpholino-injected embryos
(Fig. 5A,C), it was strongly
reduced in FoxF1 knockdown embryos
(Fig. 5B,D). Counting of
BrdU-positive cells revealed that the lateral plate mesoderm of FoxF1
knockdown tadpoles contained about 40% less BrdU-positive cells
(Fig. 5E). At the same time the
cell proliferation in the neuroectoderm was not affected. This demonstrates a
significant reduction of cell proliferation in the lateral plate mesoderm of
FoxF1 morpholino-injected embryos.
|
Regulation of FoxF1 during Xenopus development
Dorsoventral properties of the mesoderm depend on antagonistic interactions
between dorsalizing and ventralizing signals
(Heasman, 1997;
Sive, 1993
). The major
ventralizing signal in Xenopus embryos is the signaling molecule
BMP4. Since FoxF1 is predominantly expressed in the ventral mesoderm,
BMP4 might an upstream regulator of FoxF1. This possibility is
further strengthened by the observation in Drosophila that
biniou is regulated by dpp, the Drosophila homolog
of BMP4 (Zaffran et al.,
2001
). We performed a series of experiments in order to address
this possibility. First, we injected BMP4 RNA into 8-cell stage
Xenopus embryos. At early blastula stage we cut animal caps and
cultured them until sibling embryos reached the late gastrula stage. We used
RTPCR to determine whether these caps expressed FoxF1 RNA. As can be
seen in Fig. 6A, the BMP4
RNA-injected caps expressed FoxF1 mRNA, while the control, uninjected
caps did not express FoxF1 mRNA. This shows that BMP4 alone can
activate FoxF1 transcription in uncommitted ectoderm.
|
In the third experiment, we examined the ability of FoxF1 RNA to
rescue dorsalized embryos that suffer from a lack of BMP4 signaling. It has
been demonstrated previously that embryos injected with the dominant negative
BMP4 receptor (DNBR) into the ventral blastomeres of 8-cell stage embryos lost
most of their ventral structures. This is because DNBR eliminates BMP4
signaling; and as a result, the ventral mesoderm converts to dorsal mesoderm,
leading to the formation of a secondary axis
(Suzuki et al., 1994). If
FoxF1 is a downstream target of BMP4 and mediates BMP4 function, a
co-injection of FoxF1 RNA with DNBR RNA should lead to a rescue of
the dorsalized phenotype and to the elimination of the secondary axis. As can
be seen in Fig. 6G,H, this was
indeed the case. Injection of DNBR RNA led to formation of the secondary axis
in 95.8% (69/72) of embryos (Fig.
6G). By contrast, when FoxF1 RNA was co-injected with the
DNBR RNA, only 4.4% (4/90) of embryos formed a secondary axis
(Fig. 6H). This provides
further evidence that FoxF1 is a downstream mediator of BMP4
signaling.
Several papers demonstrated induction of mesodermal markers in
Xenopus animal caps by BMP4. For example, Jones et al.
(Jones et al., 1992) and
Suzuki et al. (Suzuki et al.,
1997
) showed that BMP4 can induce transcription of
mesoderm-specific genes such as Brachyury (Xbra). Since our
experiments confirmed these findings (Fig.
6A), we examined the possibility that the activation of
FoxF1 might be mediated by Xbra. Xbra has a partially
overlapping pattern of expression during early Xenopus development
and could be a potential mediator of BMP4 signaling in FoxF1
activation. Indeed, our experiments showed that Xbra could activate
FoxF1 in animal caps (Fig.
6B), opening the possibility that FoxF1 activation by
BMP4 is indirect.
Furthermore, since the lack of gut coiling in Xenopus embryos
injected with FoxF1 morpholinos strongly resembles the phenotype of
embryos injected with RNA encoding the dominant negative FGF receptor 1
(Saint-Jeannet et al., 1994),
we examined whether FGF can also activate this gene. As can be seen in
Fig. 6B, FoxF1 could
be activated in animal caps by FGF as well. All these experiments suggest that
the regulation of FoxF1 expression is complex and that there might be
more than one way of activating this gene. In order to shed more light on the
regulation of FoxF1, we decided to identify the cis-regulatory sequences that
are responsible for the correct temporal-spatial expression of this gene.
FoxF1 regulatory elements are located in the 5' upstream region of the gene
Since the FoxF1 gene must contain regulatory sequences that direct
gene expression into the lateral plate mesoderm, we used transgenic frog
embryos to identify the segment of FoxF1 DNA that is responsible for
the temporal-spatial regulation of FoxF1 expression. For this
purpose, we isolated a genomic clone of FoxF1 and, using GFP or LacZ
as a reporter, we delineated the sequences that can direct gene expression
into the lateral plate mesoderm. We found that a 2 kb DNA fragment upstream of
the 5' end of the transcription start site of FoxF1 can direct
GFP expression into the lateral plate mesoderm of Xenopus tadpoles
(Fig. 7A-C). When performing
these experiments, we found that while the monitoring of the GFP fluorescence
was informative, it was difficult to evaluate GFP expression in sections as
the autofluorescence of the yolk made the evaluation of GFP fluorescence
unreliable. For this reason, we performed whole-mount in-situ hybridization on
transgenic embryos with an anti-GFP digoxigenin-labeled probe. This eliminated
the problem with autofluorescence and allowed visualization of GFP expression
in sections (Fig. 7D,E). The
levels of GFP RNA varied, but in general they were limited to the lateral
plate mesoderm (Fig. 7D). In a
significant portion of embryos, the expression was only on one side of the
embryo (Fig. 7E). We are
currently investigating whether this expression pattern is due to a delayed
integration of the transgene or due to the fact that the transgene contains
sequences that bias gene expression toward unilateral expression. To further
refine this promoter, we also used LacZ as a reporter system. The expression
pattern of LacZ was similar to GFP, but the expression was somewhat more
mosaic (Fig. 7G). However, the
appearance of mosaicism is enhanced by the fact that the nuclear localization
signal of the LacZ reporter construct (Fig.
7F) directs the protein into the nuclei. Nevertheless, all these
experiments show that the 2 kb upstream fragment of the FoxF1
contained the necessary sequences to direct gene expression into the
developing lateral plate mesoderm. Since we have shown in previous experiments
that FoxF1 might be regulated by BMP4 signaling, we investigated
whether this LacZ reporter construct is responsive to BMP4 induction. As can
be seen in Fig. 7I, animal caps
injected with this construct and BMP4 RNA display significant expression of
the LacZ reporter. This is in contrast to the animal caps injected with the
construct alone (Fig. 7H). This
shows that the 2 kb construct contains sequences that mediate BMP4
signaling.
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Discussion |
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At high concentrations of FoxF1 morpholino, the visceral mesoderm surrounding the gut loses its physical integrity completely and the yolky endodermal cells spill into the body cavity. Eventually the entire ventral body cavity will lyse, which might indicate that the somatic mesoderm is also deficient in cell numbers as a result of reduced proliferation in the lateral plate mesoderm during neurulation.
When one compares Xenopus FoxF1 with its murine ortholog, several
similarities can be found. In addition to the structural conservation between
these two genes, Xenopus and murine Foxf1 have a similar
expression pattern. In both species Foxf1 is expressed in the lateral
plate/visceral mesoderm. In the mouse, additional high levels of
Foxf1 transcription can be found in the extra-embryonic
mesoderm-derived structures -amnion, allantois and yolk sack - which are not
found in Xenopus. These two genes also show high levels of functional
conservation. In both species the formation of visceral mesoderm is strongly
affected by the expression of FoxF1. A lower rate of proliferation is
present in the lateral plate mesoderm of FoxF1 morpholino-treated
Xenopus embryos as well as in Foxf1-/- mouse embryos
(Mahlapuu et al., 2001b).
Unfortunately, the Foxf1-/- mice die and degenerate before gut
organogenesis begins, and therefore they do not provide us with information
about the role of Foxf1 in the murine gut development. The
haploinsufficient Foxf1+/- mice, which presumably have 50% of the
Foxf1 activity intact, suffer from high perinatal mortality, partly
caused by constriction of the esophagus, and provide information limited to
foregut malformations (Mahlapuu et al.,
2001a
). However, the abnormalities observed in development of the
allantois in Foxf1 knockout mouse embryos are in many respects
similar to those present in the development of Xenopus gut mesoderm.
Allantois is a mesodermal structure that during normal development elongates
and fuses with the chorion. In Foxf1-/- embryos, allantois does not
differentiate and expands properly, and consequently does not reach the
chorion. A proper placentation does not take place and the mutant embryos die
(Mahlapuu et al., 2001b
).
While Foxf1 is the best-studied Fox gene in the development of the
visceral mesoderm, a functional loss of the Fox gene Foxl1
(Fkh6) has also been shown to result in detrimental effects on the
development of the gastrointestinal epithelium
(Kaestner et al., 1997).
Foxl1 is expressed in the visceral mesoderm and its function is
required for regulating normal gastrointestinal proliferation and
differentiation. Foxl1-/- mice show severe abnormalities of gut
development including changes in the endoderm-derived gut epithelium.
Therefore the likely role of Foxl1 resides in the interaction between
mesenchyme and epithelial cells. The putative Xenopus ortholog of
Foxl1, FoxL1 (XFD-8), has been isolated, but no expression of this
gene was detected in embryonic stages (Lef
et al., 1996
). This might be because FoxL1 has no
function in embryonic development or because low levels of expression make the
detection of its transcripts difficult.
Interestingly, the critical role of the FoxF gene family in the development
of visceral mesoderm extends also to invertebrates. The Drosophila
gene biniou (FoxF), which is the fly homolog of
FoxF1, has an important function in the development of the
Drosophila midgut (Zaffran et
al., 2001). bin is activated in the Drosophila
trunk visceral mesoderm primordia and has a critical role during specification
and differentiation of the trunk visceral mesoderm. In its absence, the
differentiation of the splanchnic mesoderm into midgut musculature is
impaired. Taken together, all these data indicate that the FoxF family is an
ancient gene family that has a conserved role in the visceral mesoderm
development in vertebrates and invertebrates.
Regulation of FoxF1
Our experiments suggest that Xenopus FoxF1 is a target and a
mediator of BMP4 signaling. Several pieces of evidence support this
conclusion. First, BMP4 RNA could induce expression of FoxF1 in
animal caps. Second, injection of FoxF1 RNA into Xenopus
embryos ventralized embryos, mimicking the action of BMP4. Finally,
FoxF1 RNA could rescue dorsalized embryos resulting from the
injection of dominant negative BMP4 receptor. This BMP4 regulation of
FoxF1 is likely to reflect an evolutionarily conserved pathway, as
biniou in the Drosophila visceral mesoderm is dependent on
dpp, the fly homolog of BMP4
(Frasch, 1995). However, it is
unlikely that the BMP4 pathway is the only avenue for regulating the
expression of FoxF1 in vertebrates. First, expression of
Foxf1 is largely unaffected in BMP4 knockout mouse embryos
(Fujiwara et al., 2002
). While
it is possible that other BMP proteins compensate for the lack of BMP4 protein
in these knockout embryos, there are several experiments suggesting that FGF
signaling is also involved in the formation of the visceral mesoderm.
Saint-Jeannet et al. (Saint-Jeannet et
al., 1994
) demonstrated that Xenopus embryos injected
with RNA encoding the dominant-negative FGF receptor1 do not develop visceral
mesoderm and do not activate
-smooth muscle actin expression. This is
very similar to our observations of FoxF1 morpholino-treated embryos.
FGF signaling is also involved in the formation of the Drosophila
visceral mesoderm, as the mesoderm-specific FGF receptor
DFR1/Heartless is required for the formation of the visceral muscles
(Shishido et al., 1997
).
FoxF1 can also be activated by Xbra. While Xbra
is expressed primarily in the notochord of neurula-stage vertebrate embryos,
during gastrulation it is expressed in the blastopore equivalent of bilateria.
In Xenopus this means that the expression is surrounding the entire
yolk plug (Wacker et al.,
2004), making it possible that Xbra is involved in the
regulation of FoxF1. Regulation of FoxF1 by Xbra
would not be entirely surprising, as the Drosophila Brachyury
(byn) is necessary for the hindgut formation (for a review, see
Lengyel and Iwaki, 2002
). It
is not clear at present to which degree these different pathways are used for
the regulation of expression of FoxF1 and its orthologs in different
species. It is likely that the relative contribution of each pathway to the
regulation of FoxF1 will be species dependent. For instance, the
evolutionary divergence of FoxF1 regulation can be demonstrated using
the example of regulatory interactions between Drosophila bin and
bap and Xenopus FoxF1 and Xbap. While the
Drosophila biniou is activated by bap, this does not appear
to be the case in Xenopus. This is because the timing of expression
of FoxF1/bin and Xbap/bap has changed during evolution.
While Drosophila bap is transcribed in the visceral mesoderm almost
simultaneously with bin (Zaffran
et al., 2001
), Xenopus bap is activated several hours
after FoxF1 and therefore cannot be involved in the initiation of
FoxF1 transcription.
Further analysis of FoxF1 regulation was facilitated by the
finding that the 2 kb upstream region of this gene is responsive to BMP4
signaling. While a computer search for potential protein-binding sites did not
detect any BMP4 responsive elements (BRE), we discovered a potential
Vent-2 binding site. Vent-1/PV.1
(Gawantka et al., 1995;
Ault et al., 1996
) and
Vent-2 (Onichtchouk et al.,
1996
) proteins belong to the group of homeodomain proteins that
mediate BMP4 signaling in the formation of ventral mesoderm. While this
finding sheds some light on the potential regulatory mechanism of BMP4
signaling, the computer analysis of this fragment also locates a potential
Brachyury-binding site. This site could account for the inducibility
of FoxF1 by Xbra. In addition, this fragment of DNA contains
potential binding sites for several transcription factors that are known to be
involved in gut formation. There are potential binding sites for GATA1-3, for
the intestine-specific homeodomain factor CDX-1 and the mammalian
caudal-related intestinal transcription factor Cdx-2 and a binding site for
HNF3beta. A detailed experimental analysis of these potential protein-binding
sites will have to be performed before a meaningful credibility can be
assigned to any of these sites.
In summary, analysis of FoxF1 genes in Drosophila,
Xenopus and mouse leads to a unified picture in which this ancient gene
was initially involved in the proliferation and differentiation of the
visceral mesoderm. In the absence of its function, normal development of the
visceral mesoderm cannot take place. In vertebrates, the lack of
FoxF1 leads to a variable phenotype depending on the peculiarities of
the development of the given species. In mammals, where this gene is involved
in the formation of extra-embryonic support structures, the lack of
FoxF1 gene product leads to early embryonic death because of the
failure of these structures to provide essential support to the embryo proper.
In Xenopus embryos, which do not have any extra-embryonic structures,
embryos display abnormal formation of smooth muscles around the gut. How
exactly FoxF1 regulates proliferation and differentiation is not
clear, but it is possible that it belongs to a group of proteins that are able
to couple these two regulatory functions, such as the Hox and Six proteins
(Del Bene et al., 2004;
Luo et al., 2004
).
Last, but not least, we have isolated the FoxF1 regulatory sequences that are able to direct gene expression into the developing lateral plate mesoderm. These sequences are located in the 5' upstream region of the gene. Isolation of sequences that specifically direct gene expression into the developing gut mesoderm will allow us specifically to alter gene expression in the gut mesoderm and monitor the morphological, physiological and molecular consequences of this altered gene expression in the gut mesoderm and endoderm. This is of extreme importance, as the lateral plate mesoderm, and later the visceral mesoderm, have an instructive role in the regionalization and proper development of the gut endoderm. This will make Xenopus uniquely suited for studying epithelial-mesenchymal interactions during gut development.
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ACKNOWLEDGMENTS |
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