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First published online 8 November 2006
doi: 10.1242/dev.02663
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Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, 1110 BRB 2/3, 421 Curie Boulevard, Philadelphia, PA 19104, USA.
Author for correspondence (e-mail:
kesslerd{at}mail.med.upenn.edu)
Accepted 28 September 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Xenopus, FoxD3, Forkhead, Nodal, Mesoderm, Transcription
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Heasman, 2006;
De Robertis et al., 2000). For
example, in Xenopus mesoderm formation, Nodal signals are subjected
to multiple positive and negative inputs that reinforce pathway activity in
the mesodermal domain and exclude pathway activity in the adjacent ectodermal
region (Schier and Shen, 2000;
Whitman, 2001).
The Fox gene family comprises a large and functionally diverse group of
forkhead-related transcriptional regulators, many of which are
essential for metazoan embryogenesis and physiology
(Carlsson and Mahlapuu, 2002;
Lehmann et al., 2003;
Pohl and Knochel, 2005). FoxD3
is a member of the Fox family that has multiple roles in the vertebrate
embryo, including regulation of neural crest development and maintenance of
mammalian stem cell lineages. FoxD3 orthologs in Xenopus
(Xfd6/Xfkh6), zebrafish (Fkd6), chick
(Cwh3) and mouse (Genesis/Hfh2) are expressed in
the neural crest (Dirksen and Jamrich,
1995; Scheucher et al.,
1995; Lef et al.,
1996; Sutton et al.,
1996; Freyaldenhoven et al.,
1997a; Labosky and Kaestner,
1998; Odenthal and
Nusslein-Volhard, 1998;
Yamagata and Noda, 1998;
Kelsh et al., 2000). Studies
in Xenopus and chick indicate that FoxD3 regulates the determination,
migration, survival and/or differentiation of a number of neural crest
lineages (Dottori et al.,
2001; Kos et al.,
2001; Pohl and Knochel,
2001; Sasai et al.,
2001; Cheung et al.,
2005; Whitlock et al.,
2005; Lister et al.,
2006; Stewart et al.,
2006). A role in the neural crest is further supported by the
association of a human FOXD3 promoter sequence variant with autosomal
dominant vitiligo, a pigmentation disorder caused by defects in the
melanoblast lineage (Alkhateeb et al.,
2005).
Foxd3 is also expressed in the preimplantation mouse embryo, in
mouse and human embryonic stem cells, and in mouse trophoblast stem cells
(Sutton et al., 1996;
Pera et al., 2000;
Hanna et al., 2002;
Tompers et al., 2005).
Foxd3 null embryos have a severe reduction of epiblast cell number
and die by 6.5 days postcoitum (dpc), and Foxd3 null trophoblast
progenitors are defective in both self-renewal and differentiation. In
addition, neither embryonic stem cell lines nor trophoblast stem cell lines
can be established from Foxd3 null embryos
(Hanna et al., 2002;
Tompers et al., 2005). The
requirement for Foxd3 in both embryonic and trophoblast stem cells
suggests that Foxd3 may also be required in multipotent neural crest
stem cells, but it is not yet known if the molecular and developmental
functions of Foxd3 are similar in these diverse progenitor populations.
Prior to expression in the neural crest, FoxD3 is expressed in the
Spemann organizer, the zebrafish shield, and the chick and mouse node (see
Fig. S1 in the supplementary material)
(Labosky and Kaestner, 1998;
Odenthal and Nusslein-Volhard,
1998; Yamagata and Noda,
1998; Pohl and Knochel,
2001; Sasai et al.,
2001; Yaklichkin et al.,
2003), the gastrula signaling center that controls germ-layer
patterning, morphogenesis and axis formation (reviewed by
Harland and Gerhart, 1997;
De Robertis et al., 2000).
Here we report that FoxD3 function in the Spemann organizer is essential for
dorsal mesodermal development. FoxD3 functions as a transcriptional repressor
to induce dorsal mesoderm and axis formation, and antagonism or knockdown of
FoxD3 results in severe axial defects and loss of dorsal mesodermal gene
expression. FoxD3 induction of mesoderm is non-cell-autonomous and requires
the Nodal signaling pathway. Consistent with the co-expression of
FoxD3 and Nodal genes in the organizer, FoxD3 is necessary
and sufficient for the expression of several Nodal-related genes.
Taken together, our results demonstrate a novel mode of Nodal regulation in
the Spemann organizer, where transcriptional repression by FoxD3 maintains
Nodal expression to promote mesoderm induction and axial
development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and embryonic stage was determined according to Nieuwkoop
and Faber (Nieuwkoop and Faber,
1967). Dorsal and ventral blastomeres were identified by
pigmentation differences (Klein,
1987). Explants were prepared using a Gastromaster microsurgery
instrument (Xenotek Engineering). Capped, in vitro transcribed RNA for
microinjection was synthesized from linearized template DNA using the Message
Machine kit (Ambion) and 10 nl of RNA solution was injected. Templates for in
vitro transcription were pCS2-FoxD3, pCS2-mFoxD3, pCS2-Eng-FoxD3,
pCS2-VP16-FoxD3, pCS2-FoxD3(N140A/H144A), pCS2-Eng-FoxD3(N140A/H144A),
pCS2-VP16-FoxD3(N140A/H144A), pCS2-NLS-FoxD3WH, pCS2-FoxD3-utr (this study),
pCS2-Eng, pCS2-VP16 (Kessler,
1997), pCS2-MT-SID (Chen et
al., 1997), pCS2-Cer-S
(Piccolo et al., 1999),
pCS2-Xnr1 (Sampath et al.,
1997), and pCS2-VegT
UTR
(Engleka et al., 2001).
FoxD3 expression constructs
The FoxD3 constructs described in this study were generated by subcloning
into pCS2+, pCS2-NLS, or pCS2-GFP (Rupp et
al., 1994). A FoxD3 cDNA clone (nucleotides 105-1308) containing
the ORF flanked by 67 nucleotides of 5'UTR and 21 nucleotides of
3'UTR was obtained by RT-PCR of tailbud stage mRNA using primers derived
from the published sequence of Xenopus FoxD3
(Dirksen and Jamrich, 1995).
This subclone, referred to in this study as pCS2-FoxD3, pCS2-xFoxD3 or
pCS2-FoxD3+utr, was used to generate the additional FoxD3 constructs. A
detailed description of the Xenopus FoxD3 constructs used in this
study is provided in Supplementary Material (see Fig. S2 in the supplementary
material). The mouse Foxd3 construct (pCS2-mFoxD3) was generated by subcloning
an EcoRI genomic fragment containing the ORF flanked by 75 nucleotides of
5'UTR and 600 nucleotides of 3'UTR
(Labosky and Kaestner,
1998).
Morpholino oligonucleotides
The FoxD3 antisense morpholino oligonucleotide (FoxD3MO) is complementary
to nucleotides 158-181 of Xenopus FoxD3
(5'-ACAGGGTCATTCCAGTTACGCTCC-3') and was injected at 10-100 ng per
embryo (Gene Tools). As a control, embryos were injected with equal doses of a
mismatch morpholino oligonucleotide (misMO) complementary to nucleotides
158-181 of FoxD3 at all but five positions
(5'-ACAcGGTgATTCaAGTTACcCTgC-3').
In situ hybridization, immunocytochemistry and histology
For whole-mount in situ hybridization, embryos were fixed and hybridized
with antisense, digoxygenin-labeled RNA probes as described
(Sive et al., 2000).
Hybridized probe was detected using alkaline phosphatase-conjugated
anti-digoxygenin Fab fragments (Boehringer-Mannheim) and BMpurple
(Boehringer-Mannheim) as substrate for color development. Antisense probes
were synthesized from linearized plasmid DNA using the Megascript kit (Ambion)
supplemented with 2 mM digoxygenin-11-UTP. Templates for in situ probes were
pGEM-Xbra (Wilson and Melton,
1994), pCS2-Chd (Sasai et al.,
1994), pBS-Dlx3 (Feledy et
al., 1999), pGEM-Gsc (Cho et
al., 1991), pT7blue-Mixer
(Engleka et al., 2001),
pBS-Opl (Kuo et al., 1998),
pBS-Xnr1, pBS-Xnr2 (Jones et al.,
1995), and pGEM-Xwnt8 (Sokol
et al., 1991). For serial section immunocytochemistry, embryos
were embedded in paraplast as described
(Sive et al., 2000), and 15
µm sections were stained with monoclonal antibodies specific for muscle
(12/101) (Kintner and Brockes,
1984), notochord (Tor70) (Bolce
et al., 1992), or neural tissue (4d)
(Watanabe et al., 1986), and
HRP-coupled secondary antibody. Positive staining was visualized with VIP,
DAB+Ni or DAB as HRP substrates (Vector Laboratories). For histology, 10 µm
sections were prepared from paraplast-embedded embryos and explants, and
dewaxed sections were stained with Hematoxylin/Eosin before coverslipping with
Permount. For double-staining, samples were processed for in situ
hybridization, and following the chromogenic reaction, samples were fixed and
processed for immunocytochemistry as previously described
(Sive et al., 2000).
Reverse transcription-polymerase chain reaction and western analysis
For RT-PCR, total RNA was isolated using the RNAqueous kit (Ambion), and
cDNA synthesis and PCR were performed as described
(Wilson and Melton, 1994).
Radiolabeled PCR products were resolved on 5% native polyacrylamide gels. PCR
primers and cycle parameters were as described for EF1
, Xbra, Xwnt8,
Muscle Actin, NCAM (Wilson and Melton,
1994), Collagen Type II (Agius
et al., 2000), MyoD (Rupp et
al., 1994), Xnr1, Xnr2
(Sampath et al., 1997), Xnr4
(Joseph and Melton, 1997) and
Derriere (Sun et al., 1999).
For western analysis, injected embryos were lysed (10 µl per embryo) in 0.1
M Tris-HCl (pH 6.8) supplemented with protease inhibitors. The extracts were
cleared by centrifugation and half an embryo equivalent was loaded per well.
An affinity-purified anti-Xenopus FoxD3 polyclonal antibody (see Fig.
S1 in the supplementary material) (this study)
(Tompers et al., 2005), was
used at a 1:1000 dilution and was detected with a 1:3000 dilution of
anti-rabbit IgG-peroxidase by chemiluminescence (Amersham). As a loading
control, stripped blots were analyzed with a monoclonal antibody against MAPK
(ERK1/2) (Sigma). For analysis of phospho-Smad2, animal explant lysates were
prepared as previously described (Lee et
al., 2001), and phospho-Smad2 was detected using a
phospho-specific monoclonal antibody (Cell Signaling). As a loading control,
stripped blots were analyzed with a polyclonal antibody against Smad2/3 (Cell
Signaling).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
notochord (Tor70) (Bolce et al.,
1992), and neural tissue (4d)
(Watanabe et al., 1986). All
affected embryos contained a mass of ectopic muscle
(Fig. 1D) and an expansion and
disorganization of the neural tube (Fig.
1F). Embryos with ectopic anterior structures displayed two, and
sometimes three, notochords (Fig.
1E). Consistent with the observed effects on axis formation,
expression of FoxD3 in ventral marginal zone explants induced markers of
dorsal mesoderm and differentiation of dorsal axial tissues (see Fig. S3 in
the supplementary material).
The influence of ectopic FoxD3 on mesodermal pattern was also examined at
the gastrula stage. At the four-cell stage, FoxD3 mRNA was injected
into a single ventral blastomere, and embryos were collected for whole-mount
in situ hybridization at the early gastrula stage. Consistent with the axial
effects, FoxD3 induced ectopic expression of Goosecoid, an organizer
marker (Fig. 1H). The results
demonstrate that FoxD3 is sufficient for ectopic dorsal mesoderm formation and
suggest a role for FoxD3 in endogenous mesoderm formation and/or patterning,
consistent with the expression of FoxD3 in the Spemann organizer. We
note that the response to FoxD3 is similar to activation of the Smad2 pathway
by TGFß-related proteins, which induce dorsal mesoderm formation, and Wnt
activation of the ßcatenin pathway, which dorsalizes ventral mesoderm
(Heasman, 2006).
|
)
(Fig. 2A-C). To confirm that
mesoderm induction had occurred, explants were analyzed by
immunocytochemistry, histology and RT-PCR. The presence of differentiated
somitic muscle was detected at the tailbud stage by whole-mount
immunocytochemistry with a muscle-specific monoclonal antibody (12/101)
(Kintner and Brockes, 1984).
Whereas control explants had no detectable muscle, nearly all FoxD3-expressing
explants (90%, n=20) contained abundant muscle
(Fig. 2D,E). The explants were
subsequently sectioned and counterstained with Hematoxylin/Eosin for
histological analysis. FoxD3-expressing explants contained somitic muscle,
notochord, and neural tissue, whereas control explants contained only ciliated
epidermis (Fig. 2G,H). Gene
expression was examined by RT-PCR at the midgastrula and tailbud stages. FoxD3
induced the expression of Brachyury (panmesodermal),
Goosecoid (dorsal mesoderm/organizer) and Xwnt8
(ventrolateral mesoderm) at the midgastrula stage. Additional organizer
markers, including Chordin and Noggin, were also induced by
FoxD3 (data not shown). At the tailbud stage, FoxD3 induced the expression of
Muscle Actin (somitic mesoderm), Lim1 and Pax8
(pronephros), and NCAM (pan-neural), but not markers of heart
(Nkx2.5 and Tbx5) or blood (AML and
T4-Globin) (Fig.
2J and data not shown). Identical results were obtained for the
Xenopus and mouse orthologs of FoxD3
(Fig. 2C,J and data not shown).
Therefore, FoxD3 is sufficient for mesodermal gene expression and the
induction of differentiated axial mesoderm. This mesoderm-inducing activity of
FoxD3 is most similar to the Smad2-activating TGFß-related ligands,
including Activin, Vg1 and Nodal (Heasman,
2006).
FoxD3 functions as a transcriptional repressor to induce mesoderm
As a member of the Forkhead family of transcriptional regulators, it is
predicted that FoxD3 induces mesoderm by transcriptional activation or
repression of specific target genes. To determine the transcriptional activity
of FoxD3 responsible for mesoderm induction, the activity of chimeric FoxD3
proteins containing the FoxD3 DNA-binding domain fused to defined
transcriptional regulatory domains was examined. In this strategy, the
specific DNA-binding domain delivers a strong activator or repressor to
endogenous target genes and stimulates or inhibits their transcription
(Conlon et al., 1996;
Kessler, 1997). Chimeric
proteins were generated containing the HSV VP16 activator domain
(Sadowski et al., 1988;
Triezenberg et al., 1988) or
the Drosophila Engrailed repressor domain
(Jaynes and O'Farrell, 1991;
Han and Manley, 1993;
Badiani et al., 1994) fused to
the winged helix DNA-binding domain of FoxD3
(Fig. 3A). The
mesoderm-inducing activities of the Engrailed repressor fusion protein
(Eng-FoxD3) and the VP16 activator fusion protein (VP16-FoxD3) were examined
by expression in animal explants. Like native FoxD3, Eng-FoxD3 induced
convergent extension movements, whereas VP16-FoxD3 did not have this effect
(Fig. 3B-E). Consistent with
the morphology of the explants, Eng-FoxD3 induced the expression of Muscle
Actin and Collagen Type II, a notochord marker, whereas
VP16-FoxD3 did not activate these axial mesoderm markers
(Fig. 3F). Histological
analysis at the tailbud stage and RT-PCR analysis at the gastrula stage
confirmed that the mesoderm-inducing activities of Eng-FoxD3 and native FoxD3
were indistinguishable (data not shown). Furthermore, like native FoxD3,
Eng-FoxD3 induced ectopic dorsal mesoderm when expressed in the ventral
marginal zone (data not shown). The results suggest that FoxD3 functions as a
transcriptional repressor to induce mesoderm. In a Gal4-UAS transcriptional
assay, FoxD3 repressed basal transcription of a luciferase reporter
15-fold in animal explants at the gastrula stage
(Yaklichkin et al., 2006).
This result confirms that FoxD3 functions as a transcriptional repressor,
consistent with previous studies of FoxD3 orthologs in cell culture
and in the neural crest lineage (Sutton et
al., 1996; Freyaldenhoven et
al., 1997b; Pohl and Knochel,
2001; Sasai et al.,
2001).
|
For the chimeric proteins, the FoxD3 DNA-binding domain is predicted to deliver the activator or repressor domains to specific target genes normally regulated by FoxD3. To confirm that DNA-binding activity is required for the function of the native and chimeric forms of FoxD3, conserved DNA contact residues were mutated (N140A/H144A) to generate DNA-binding inactive forms of native FoxD3 and the chimeric proteins (see Fig. S2 in the supplementary material). In animal explants the DNA-binding inactive forms of FoxD3 and Eng-FoxD3 did not induce mesoderm, and the VP16-FoxD3 mutant did not inhibit the activity of native FoxD3 (data not shown). In addition, the individual domains that comprise the chimeric FoxD3 proteins (FoxD3 DNA-binding domain, VP16 activator and Engrailed repressor) had no activity (data not shown). Therefore, sequence-specific DNA-binding activity is required for the function of native and chimeric forms of FoxD3.
Taken together, the results indicate that FoxD3 functions as a transcriptional repressor to induce mesoderm. Beyond defining the transcriptional activity of FoxD3 responsible for mesoderm induction, the results have an unexpected implication for the regulation of mesodermal development. The ability of FoxD3 and Eng-FoxD3 to induce mesoderm argues for the presence of a negative regulator of mesoderm formation that is repressed by FoxD3. This suggests that the establishment of mesoderm in Xenopus may involve transcriptional repression of a mesodermal inhibitor.
FoxD3 is required for axial and mesodermal development
Loss-of-function analysis can be accomplished in Xenopus by
injection of an antisense morpholino oligonucleotide (MO) that specifically
blocks translation of a target mRNA
(Summerton and Weller, 1997;
Heasman et al., 2000). To
determine the requirement for FoxD3 function in Xenopus
mesodermal development, a MO was designed that is complementary to the
FoxD3 mRNA in the region of the initiator methionine codon
(Fig. 4A). FoxD3MO is predicted
to form a stable heteroduplex with FoxD3 mRNA and block translational
initiation (Summerton and Weller,
1997). To assess the efficacy of FoxD3MO, embryos were injected
with FoxD3 mRNA and FoxD3MO or a control MO containing five
mismatches with the FoxD3 target sequence (mismatch MO), and FoxD3 translation
in animal explants was examined by western blot analysis
(Fig. 4B). Translation of a
FoxD3 RNA containing the entire target sequence (FoxD3+utr) was
blocked by FoxD3MO, whereas a FoxD3 RNA lacking the 5'UTR
target sequence (FoxD3-utr) was translated normally. The mismatch MO did not
inhibit the translation of either FoxD3 RNA. The ability of FoxD3MO
to interfere with the mesoderm-inducing activity of FoxD3 was examined in
animal explants. Consistent with the observed translational block, FoxD3MO
inhibited the induction of Muscle Actin by FoxD3+utr, but did not
affect the response to FoxD3-utr (Fig.
4C). Mismatch MO did not block induction by either RNA. To assess
the ability of FoxD3MO to inhibit translation of endogenous FoxD3, embryos
injected with FoxD3MO or mismatch MO were analyzed by western blotting at the
midgastrula stage (Fig. 4D). A
single major protein identical in size to overexpressed Xenopus FoxD3
was detected in uninjected and mismatch MO-injected embryos, and FoxD3MO
resulted in an tenfold reduction in protein levels. This striking
inhibition of endogenous FoxD3 translation suggests that FoxD3MO injection
results in a complete or near complete loss-of-function for FoxD3.
|
The inhibition of axis formation by VP16-FoxD3 and FoxD3MO is predicted to result from a specific block of endogenous FoxD3 function. To determine the specificity of FoxD3 inhibition, FoxD3 was co-injected with VP16-FoxD3 or FoxD3MO in an attempt to rescue axis formation (Table 1 and Fig. S4 in the supplementary material). Whereas the majority of VP16-FoxD3-injected embryos had severe axial defects (73%, n=44), only a minority displayed defects with FoxD3 co-injection (13%, n=61). Similarly, the axial defects caused by FoxD3MO (79%, n=38) were rescued by FoxD3 RNA lacking the antisense target sequence (FoxD3-utr) (9%, n=54), but not by FoxD3 RNA containing the target sequence (FoxD3+utr) (67%, n=49). As controls, injection of both dorsal blastomeres with FoxD3 RNA or mismatch MO did not perturb axis formation. The rescue of axis formation by FoxD3 indicates that VP16-FoxD3 and FoxD3MO are specific inhibitors of endogenous FoxD3.
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Mesoderm induction by FoxD3 is non-cell-autonomous and dependent on Nodal signaling
The mesoderm-inducing activity of FoxD3 is identical to Smad2-activating
members of the TGFß family, including the Nodal-related
genes required for mesoderm formation
(Heasman, 2006;
Schier and Shen, 2000). This
suggested that FoxD3 may interact with a Smad2-activating pathway to induce
mesoderm, either as an upstream regulator of ligand expression, or as a
downstream mediator of the response to active Smad2. To assess the potential
involvement of secreted factors in the response to FoxD3, the cell autonomy of
mesoderm induction by FoxD3 was examined in dissociated animal explants. In
this approach, explants prepared before the midblastula transition are
dissociated into individual cells in calcium-free medium to prevent a response
to zygotically expressed secreted factors
(Sargent et al., 1986;
Wilson and Melton, 1994).
Control and FoxD3-expressing animal explants were prepared at the early
blastula stage (stage 7), and intact or dissociated explants were examined for
mesodermal gene expression at the gastrula stage. In intact explants, FoxD3
induced expression of Brachyury and MyoD, but mesodermal
gene expression was not observed in dissociated explants
(Fig. 6A). To further assess
the autonomy of FoxD3 function in mesoderm induction, FoxD3 RNA was injected
into a single animal pole blastomere at the 32-cell stage, and the
distribution of mesodermal gene expression and FoxD3 protein was examined in
gastrula explants (Fig. 6B).
Brachyury expression was induced in a ring of cells adjacent to, but
not overlapping a group of cells containing nuclear FoxD3 protein.
Brachyury mRNA and FoxD3 protein were not observed in explants of
uninjected embryos (data not shown). The results indicate that FoxD3 induces
mesoderm in a non-cell-autonomous manner, consistent with a role for secreted
proteins in the response to FoxD3.
|
|
). FoxD3
induction of Brachyury at the gastrula stage and of Muscle
Actin at the tailbud stage was completely blocked by SID
(Fig. 6C), indicating a
requirement for a Smad2 pathway in the mesodermal response to FoxD3. The
requirement for Nodal-related ligands was examined using a truncated form of
Cerberus (Cerberus-short; CerS) that specifically inhibits the Nodal ligands
Xnr1, Xnr2, Xnr4, Xnr5 and Xnr6 (Piccolo
et al., 1999; Agius et al.,
2000; Takahashi et al.,
2000). Co-expression of FoxD3 and CerS resulted in a substantial
reduction of Brachyury and a complete block of Muscle Actin,
demonstrating that Nodal-related signals are required for the
mesoderm-inducing activity of FoxD3 (Fig.
6D). The residual Brachyury expression suggests that
there may be additional, CerS-insensitive activators of Smad2 that act
together with Nodal proteins to mediate the response to FoxD3. As a positive
control for inhibitory activity, SID and CerS blocked the mesoderm-inducing
activity of Xnr1 (Fig. 6C,D).
The results suggest that FoxD3 acts via secreted Nodal-related ligands and a
Smad2 signaling pathway to induce mesoderm. Moreover, the defects in axis and
mesoderm formation resulting from VP16-FoxD3 and FoxD3MO are consistent with a
loss of Nodal function (Osada and
Wright, 1999; Piccolo et al.,
1999; Agius et al.,
2000).
|
;
Joseph and Melton, 1997;
Agius et al., 2000). At the
gastrula stage, FoxD3 is co-expressed with Nodal-related
genes in the dorsal marginal zone (Pohl
and Knochel, 2001; Sasai et
al., 2001) (data not shown), suggesting that FoxD3 may regulate
Nodal gene expression in this dorsal mesodermal domain. To assess the
role of FoxD3 in regulating Nodal-related genes, the consequences of
FoxD3 gain-of-function and knockdown on Xnr1 and Xnr2
expression were examined at the gastrula stage by in situ hybridization.
Injection of ventral blastomeres with FoxD3 RNA induced ectopic
expression of both Xnr1 and Xnr2, demonstrating that FoxD3
can promote Nodal-related gene expression
(Fig. 7C,D). Injection of
VP16-FoxD3 or FoxD3MO resulted in a loss of Xnr1 and Xnr2
expression in the dorsal marginal zone, indicating that FoxD3 function is
required for mesodermal expression of these Nodal-related genes
(Fig. 7E-H). Interestingly,
vegetal expression of the Nodal-related genes, most apparent for
Xnr2 in these experiments, was unaffected by VP16-FoxD3 or FoxD3MO,
suggesting that FoxD3 is not required for the vegetal endodermal expression
domain (Fig. 7F,H). This result
is consistent with the unperturbed expression of Mixer and
Sox17, Nodal-responsive genes, in the vegetal domain of embryos
injected with VP16-FoxD3 or FoxD3MO (see
Fig. 5J,P and data not shown).
The mismatch MO had no effect on the marginal or vegetal expression of
Xnr1 and Xnr2 (Fig.
7I,J).
|
). FoxD3 did
not induce the expression of Xnr5 or Xnr6 (data not shown).
To determine if FoxD3 induction of Nodal expression resulted in active
signaling, phosphorylation of Smad2 was examined. In animal explants, FoxD3
induced Smad2 phosphorylation, similar to the activation of Smad2 in response
to Xnr1 (Fig. 7L). Therefore,
FoxD3 is necessary for the expression of Nodal-related genes in the
organizer, and is sufficient for the induction of Nodal-related genes
and active Nodal signaling, consistent with the embryonic defects observed
with FoxD3 knockdown.
The regulation of Nodal-related genes by FoxD3 and the dependence
of FoxD3 mesoderm-inducing activity on Nodal function suggests that
Nodal-related genes may act downstream of FoxD3 to mediate mesoderm
induction. To determine if Nodal-related genes function downstream of
FoxD3 in the dorsal marginal zone, we attempted to rescue the axial defects
resulting from FoxD3 knockdown with Xnr1. At the four-cell stage, both dorsal
blastomeres were injected with FoxD3MO alone, or in combination with
Xnr1 RNA. Whereas most embryos were affected by injection of FoxD3MO
alone (75%, n=24), co-injection of FoxD3MO and Xnr1 resulted
in a substantially reduced frequency of axial defects (24%, n=21)
(Fig. 8A-E). We note that at
the dose used, injection of Xnr1 alone resulted in anterior axial
defects in a minority of embryos (13%, n=23) (data not shown),
consistent with previous work (Piccolo et
al., 1999). In contrast to the rescue activity of Xnr1, Chordin
and Dickkopf, organizer factors that regulate axis formation by inhibition of
the BMP and Wnt pathways (Piccolo et al.,
1996; Glinka et al.,
1998), were unable to rescue FoxD3 knockdown embryos (data not
shown). The interaction of FoxD3 with Xnr1 and VegT, a direct activator of
Nodal expression (Kofron et al.,
1999; Hyde and Old,
2000), was also examined in animal explants. Xnr1 was expressed in
explants alone or in combination with FoxD3MO, and the induction of
Brachyury, MyoD, Goosecoid, Xnr1 and Xnr2 was assessed
(Fig. 8F). Mesoderm induction
and Nodal autoregulation by Xnr1 was unaffected by FoxD3MO. Similarly, VegT
induction of mesodermal and Nodal genes was unaffected by FoxD3MO. As
controls, FoxD3MO inhibited the induction of mesodermal and Nodal genes by
FoxD3, and the mismatch MO had no effect on the response to FoxD3, Xnr1 or
VegT. The observation that FoxD3 knockdown did not inhibit the activity of
Xnr1 or VegT supports a role for FoxD3 as an upstream regulator of
Nodal-related genes.
|
DISCUSSION |
|---|
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Kimelman and Bjornson, 2004).
We have identified FoxD3 as an essential regulator of dorsal mesoderm
formation. FoxD3 induces ectopic dorsal mesoderm and axis formation when
expressed outside the Spemann organizer, and FoxD3 knockdown results in
profound defects in mesodermal development and axis formation. FoxD3 is
required for the expression of multiple Nodal-related genes in the
organizer, and mesoderm induction by FoxD3 is dependent on downstream function
of the Nodal signaling pathway. FoxD3 functions as a transcriptional repressor
to induce Nodal expression and mesoderm formation, suggesting an
indirect mechanism in which FoxD3 represses target gene expression to promote
mesodermal development. Thus, we have identified FoxD3 as a novel regulator of
mesoderm formation that prevents target gene expression in the organizer. We
propose that FoxD3 functions in the Spemann organizer to repress a negative
regulator of mesodermal development and maintain the expression of
Nodal-related genes in the Xenopus gastrula.
|
; Sasai et al.,
2001; Yaklichkin et al.,
2003). Furthermore, like other organizer genes, the initiation of
Nodal-related gene expression in the organizer is dependent on
maternal VegT and nuclear ßcatenin
(Clements et al., 1999;
Kofron et al., 1999;
Agius et al., 2000;
Hyde and Old, 2000;
Lee et al., 2001;
Xanthos et al., 2002). Taken
together, the ability of regulatory inputs distinct from FoxD3 to control the
onset of endogenous Nodal expression in the organizer and the
temporal relation of FoxD3 and Nodal expression suggest that
FoxD3 likely functions to maintain, rather than initiate, Nodal
expression in the organizer following the start of gastrulation.
The activity of FoxD3 fusion proteins containing a strong activation or
repression domain indicates that FoxD3 functions as a transcriptional
repressor to induce mesoderm. This conclusion is consistent with previous
studies in cell culture and the neural crest demonstrating the repression
function of FoxD3 (Sutton et al.,
1996; Freyaldenhoven et al.,
1997b; Pohl and Knochel,
2001; Sasai et al.,
2001), and with the ability of FoxD3 to recruit Groucho
co-repressors and strongly repress reporter gene transcription
(Yaklichkin et al., 2006).
The results support a model in which FoxD3 functions as an indirect activator
of Nodal expression by repressing a negative regulator(s) of
Nodal in the organizer. The Nodal signaling pathway is essential for
multiple aspects of vertebrate development, including induction of the
endodermal and mesodermal germ layers, anterior-posterior patterning of the
body axis, and establishment of left-right asymmetry
(Schier and Shen, 2000;
Whitman, 2001). Given these
distinct roles of Nodal, it is essential that the distribution and activity of
Nodal ligand, as well as the cellular response to Nodal, be precisely
regulated. Misregulation of Nodal activity can result in gastrulation defects,
expansion of mesodermal lineages into the ectodermal domain, loss of head
structures, and situs inversus. Furthermore, as the Nodal positive feedback
loop can amplify Nodal expression and signaling, mechanisms that negatively
regulate Nodal expression and activity are essential for normal
development.
Multiple Nodal antagonists have been identified that act at each step of
the Nodal signal transduction cascade; Cerberus, Coco and Lefty/Antivin block
Nodal signaling at the extracellular level
(Thisse and Thisse, 1999;
Piccolo et al., 1999;
Cheng et al., 2000;
Bell et al., 2003;
Branford and Yost, 2004),
whereas Dapper2, Smad7, Ectodermin and PIASy act intracellularly by
stimulating receptor turnover or inhibiting Smad function
(Nakao et al., 1997;
Casellas and Brivanlou, 1998;
Daniels et al., 2004;
Zhang et al., 2004b;
Dupont et al., 2005). The
nuclear factors Drap1, Sox3, Xema and Zic2 inhibit the expression of
Nodal-related genes or the transcriptional response to Nodal signals
(Iratni et al., 2002;
Zhang et al., 2004a;
Houston and Wylie, 2005;
Suri et al., 2005). These
Nodal antagonists are functional in the Xenopus gastrula during the
period of mesoderm induction and patterning, and are thus potential regulatory
targets of FoxD3.
Therefore, FoxD3 may repress antagonists that inhibit Nodal ligand-receptor
interaction, inhibitors of Nodal signal transduction components, or repressors
of Nodal transcription. Although none of these potential mechanisms
can be excluded at this point, we favor a role for FoxD3 in repressing a
repressor of Nodal transcription. If FoxD3 were acting to relieve
inhibition of Nodal ligand or signaling components, it is predicted that
increased Nodal signaling activity would result in increased Nodal
transcription by positive feedback. However, inhibition of Nodal ligand or
signaling components, in the absence of FoxD3, would not preclude
Nodal transcription and translation, and one might expect the
accumulation of Nodal transcripts and protein. No Nodal
transcripts or active Nodal signaling is detected in the animal pole
(Jones et al., 1995;
Joseph and Melton, 1997;
Faure et al., 2000),
suggesting that Nodal genes are maintained in an `off state' and that
FoxD3 represses target genes that are required to keep Nodal
transcriptionally silent. When ectopically expressed in the animal pole, FoxD3
is predicted to derepress Nodal transcription and result in robust
Nodal expression and signaling by positive feedback. This proposed
mechanism is supported by preliminary analysis of FoxD3 regulation of the
Xnr1 promoter. Basal level transcription of an Xnr1 reporter
is strongly enhanced in response to FoxD3, suggesting that FoxD3 can
indirectly activate Nodal transcription (Q.L. and D.S.K.,
unpublished).
FoxD and mesodermal development in primitive chordates
In the primitive chordates Ciona intestinalis (ascidian) and
Branchiostoma floridae (amphioxus), a single gene homologous to the
vertebrate FoxD subfamily has been identified. Amphioxus
FoxD is expressed in the dorsal mesendoderm during gastrulation, and
is maintained in the axial mesendoderm and in the differentiating notochord
and somites. In the amphioxus gastrula there is a striking co-expression of
FoxD and Nodal in the dorsal mesendoderm (Yu et al.,
2002a,
2002b). Ciona FoxD
is expressed in the endoderm adjacent to the prospective mesoderm, and
knockdown analysis indicates that FoxD is essential for the induction of
mesodermal gene expression and notochord, but not for endodermal development
(Imai et al., 2002). In
addition, gene expression profiling of knockdown embryos indicates that FoxD
is a regulator of Nodal expression in Ciona
(Imai et al., 2004). These
observations suggest a conserved role for FoxD/FoxD3 genes in
mesodermal development of primitive chordates and vertebrates, and this may
represent the primordial developmental function for FoxD genes. We
note that amphioxus and Ciona FoxD proteins contain a heptapeptide
sequence nearly identical to the Groucho-interaction motif found in vertebrate
FoxD3 proteins (Yaklichkin et al.,
2006), suggesting a conservation of molecular, as well as
developmental function.
A Foxd3-Nodal connection in stem cell maintenance?
Foxd3 is expressed in the pre-implantation mouse embryo, in mouse
and human embryonic stem (ES) cells, and in mouse trophoblast stem (TS) cells
(Sutton et al., 1996;
Pera et al., 2000;
Hanna et al., 2002;
Tompers et al., 2005). At the
gastrula stage, Foxd3 is expressed uniformly in the epiblast,
including cells of the node, and in scattered cells of the extra-embryonic
ectoderm. Foxd3 null embryos die at 6.5 dpc with a loss of epiblast
cells and an expansion of extra-embryonic tissues. Null embryos do not
initiate gastrulation, fail to form mesoderm, and do not express
Nodal in the epiblast, but due to the early epiblast defect it is not
yet clear if FoxD3 is specifically required for mesoderm formation in the
mouse. In chimeras, a small contribution of wild-type cells can rescue null
embryos, suggesting that FoxD3 function in the epiblast is
non-cell-autonomous. In culture, the inner cell mass of null embryos initially
proliferates but is not maintained, and FoxD3 null ES cell lines cannot be
established (Hanna et al.,
2002). FoxD3 is also essential for normal placental
development, and the trophoblast progenitors of null embryos do not self-renew
and are not multipotent (Tompers et al.,
2005).
The interaction of FoxD3 and Nodal in Xenopus mesoderm formation
raises the possibility that there is an interaction between FoxD3 and Nodal in
stem cell maintenance. In fact, Nodal is required to maintain the TS
cell compartment in the mouse embryo, and Nodal protein maintains the
pluripotency of human ES cells in culture
(Besser, 2004;
Guzman-Ayala et al., 2004;
Vallier et al., 2004,
2005;
James et al., 2005). These
results suggest that Nodal, like FoxD3, is essential for
stem cell maintenance. However, Nodal null ES cell lines can be
established at expected frequencies, arguing against a requirement for Nodal
function in maintaining mouse ES cells
(Conlon et al., 1991). These
apparently contradictory results may reflect the ability of Nodal protein to
mimic a distinct TGFß ligand or, alternatively, that Nodal may function
redundantly with other TGFß ligands to maintain ES cells. Two additional
TGFß family members, Gdf1 and Gdf3, are expressed in
the early mouse embryo before or just after implantation, and both are
identical to Nodal in signaling activity
(Jones et al., 1992;
McPherron and Lee, 1993;
Rankin et al., 2000;
Cheng et al., 2003;
Chen et al., 2006;
Levine and Brivanlou, 2006).
Genetic analyses have demonstrated a synergistic interaction between
Nodal and Gdf1 in early mouse development
(Andersson et al., 2006).
Gdf3 is expressed in mouse and human ES cells and maintains markers
of pluripotency in cultured ES cells
(Clark et al., 2004;
Levine and Brivanlou, 2006).
Whether Nodal, Gdf1 and Gdf3 contribute to ES cell
maintenance in vivo, and whether FoxD3 functionally interacts with
these putative maintenance factors are significant questions for further
study.
FoxD3 has a demonstrated role in multiple processes of vertebrate development. Among the many remaining questions to explore, it will be important to identify the transcriptional targets of FoxD3 that mediate its distinct embryonic functions. Whether similar sets of FoxD3 target genes are identified in different contexts will reveal if a common regulatory pathway is utilized in each of these lineages, or if there are lineage-specific mechanisms of FoxD3 function. Ongoing studies of FoxD3 in the organizer, the neural crest, and stem cell populations are likely to provide further insight into the developmental and molecular mechanisms of vertebrate embryogenesis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/24/4827/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Agius, E., Oelgeschlager, M., Wessely, O., Kemp, C. and De Robertis, E. M. (2000). Endodermal Nodal-related signals and mesoderm induction in Xenopus. Development 127,1173 -1183.[Abstract]
Alkhateeb, A., Fain, P. R. and Spritz, R. A. (2005). Candidate functional promoter variant in the FOXD3 melanoblast developmental regulator gene in autosomal dominant vitiligo. J. Invest. Dermatol. 125,388 -391.[Medline]
Andersson, O., Reissmann, E., Jornvall, H. and Ibanez, C. F. (2006). Synergistic interaction between Gdf1 and Nodal during anterior axis development. Dev. Biol. 293,370 -381.[CrossRef][Medline]
Badiani, P., Corbella, P., Kioussis, D., Marvel, J. and Weston,
K. (1994). Dominant interfering alleles define a role for
c-Myb in T-cell development. Genes Dev.
8, 770-782.
Bell, E., Munoz-Sanjuan, I., Altmann, C. R., Vonica, A. and
Brivanlou, A. H. (2003). Cell fate specification and
competence by Coco, a maternal BMP, TGF-beta and Wnt inhibitor.
Development 130,1381
-1389.
Besser, D. (2004). Expression of nodal,
lefty-A, and lefty-B in undifferentiated human embryonic stem cells requires
activation of Smad2/3. J. Biol. Chem.
279,45076
-45084.
Bolce, M. E., Hemmati-Brivanlou, A., Kushner, P. D. and Harland, R. M. (1992). Ventral ectoderm of Xenopus forms neural tissue, including hindbrain, in response to activin. Development 115,681 -688.[Abstract]
Branford, W. W. and Yost, H. J. (2004). Nodal signaling: Cryptic Lefty mechanism of antagonism decoded. Curr. Biol. 14,R341 -R343.[CrossRef][Medline]
Carlsson, P. and Mahlapuu, M. (2002). Forkhead transcription factors: key players in development and metabolism. Dev. Biol. 250,1 -23.[CrossRef][Medline]
Casellas, R. and Brivanlou, A. H. (1998). Xenopus Smad7 inhibits both the activin and BMP pathways and acts as a neural inducer. Dev. Biol. 198, 1-12.[CrossRef][Medline]
Chen, C., Ware, S. M., Sato, A., Houston-Hawkins, D. E., Habas, R., Matzuk, M. M., Shen, M. M. and Brown, C. W. (2006). The Vg1-related protein Gdf3 acts in a Nodal signaling pathway in the pre-
