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First published online December 20, 2005
doi: 10.1242/10.1242/dev.02210
1 Center for Advanced Biotechnology and Medicine and Department of Pediatrics,
University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical
School, Piscataway, NJ 08854, USA.
2 Department of Pediatrics, Cincinnati Children's Hospital Medical Center and
University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.
3 Department of Biochemistry, University of Medicine and Dentistry of New
Jersey-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
4 Department of Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX 77030, USA.
5 Department of Pathology, Baylor College of Medicine, Houston, TX 77030,
USA.
6 Department of Molecular and Cellular Biology, Baylor College of Medicine,
Houston, TX 77030, USA.
7 Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030,
USA.
Authors for correspondence (e-mail:
mshen{at}cabm.rutgers.edu;
cbrown{at}bcm.tmc.edu)
Accepted 11 November 2005
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS Cell
RESULTS
DISCUSSION
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Key words: Tgfß signaling, EGF-CFC proteins, Anterior visceral endoderm, Mesendoderm formation
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INTRODUCTION |
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SUMMARY
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MATERIALS AND METHODS Cell
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DISCUSSION
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;
Rossant and Tam, 2004). The
AVE is formed in the most distal region of the post-implantation egg cylinder
at 5.5 days post coitum (dpc), but then translocates to the prospective
anterior side by 6.0 dpc (Rivera-Perez et
al., 2003; Srinivas et al.,
2004; Thomas et al.,
1998), prior to the onset of gastrulation. Both formation as well
as movement of the AVE require activity of the Nodal signaling pathway,
whereas the AVE itself produces Nodal antagonists that are essential for
patterning of the anterior epiblast
(Brennan et al., 2001;
Ding et al., 1998;
Perea-Gomez et al., 2002;
Yamamoto et al., 2004). As a
consequence, anti-Nodal signals from the distal visceral endoderm become
opposed to Nodal signals from the proximal epiblast, and together pattern the
anteroposterior (AP) axis following movement of the AVE. The molecular
regulation of AVE induction and Nodal signaling activities is therefore of
central importance for AP axis patterning in the mammalian embryo.
However, despite many recent advances in understanding early embryo
patterning, the function of one of the first Tgfß superfamily members to
be implicated in vertebrate development has remained poorly understood. The
Xenopus Vg1 gene was originally identified in a screen for vegetally
localized maternal transcripts in the blastula stage embryo, and was proposed
to act as an axial mesoderm inducer
(Kessler and Melton, 1995;
Melton, 1987;
Thomsen and Melton, 1993;
Weeks and Melton, 1987).
Although subsequent analyses have identified Vg1 orthologs in
zebrafish (Dohrmann et al.,
1996; Helde and Grunwald,
1993) and chick (Seleiro et
al., 1996; Shah et al.,
1997), no clear ortholog has been identified in mammalian genomes.
An initial screen for Vg1-related genes in mice had isolated
growth-differentiation factor 3 (Gdf3), which shares 79% amino acid
similarity with Vg1 (Jones et al.,
1992). Early phylogenetic comparisons had suggested that Gdf3 was
most closely related to Xenopus Vg1 and mammalian Gdf1
(Burt and Law, 1994), but more
recent analyses have grouped Gdf3 with members of the Bmp subfamily (e.g.
Chang et al., 2002;
Newfeld et al., 1999).
Several lines of evidence have suggested that Vg1 can signal through a
pathway similar to that for Nodal, which plays crucial roles in AP patterning,
formation of mesoderm, definitive endoderm and axial mesendoderm, and
specification of the left-right axis (reviewed by
Schier, 2003;
Schier and Shen, 2000;
Whitman, 2001). In particular,
signaling by processed Vg1, like that of activin and Nodal, results in
activation of Smad2, and can be antagonized by dominant-negative Smad2 mutants
(Graff et al., 1996;
Hoodless et al., 1999). In
addition, the effects of Vg1 overexpression in Xenopus can be blocked
by a dominant-negative form of Foxh1/FAST, a winged-helix transcription factor
that mediates many key aspects of Nodal signaling
(Watanabe and Whitman, 1999).
Notably, recent work has shown that the effects of Vg1 and Gdf1 overexpression
in zebrafish require the EGF-CFC protein Oep, and that mature Vg1 and Gdf1
proteins can interact with Oep as well as the mouse EGF-CFC protein Cripto
(Tdgf1) (Cheng et al., 2003).
This requirement for EGF-CFC proteins, which act as putative co-receptors for
Nodal ligand (Gritsman et al.,
1999; Reissmann et al.,
2001; Yan et al.,
2002; Yeo and Whitman,
2001), indicates that Vg1 and Gdf1 signal through a Nodal-like
pathway.
Owing to the similar activities of Vg1 and Nodal, it has remained unclear
whether there is a conservation of Vg1-related activities during mammalian
development, or indeed whether endogenous Vg1 has essential functions. To
address these issues, we have investigated the potential conservation of Vg1
function through analyses of mouse Gdf3. We find that Gdf3 has
activity in a cell culture assay for Nodal signaling, can form a complex with
activin receptors and the EGF-CFC protein Cripto, and has activities similar
to that of Nodal in Xenopus embryos and animal caps. We show that
Gdf3 is expressed in pre-implantation and early post-implantation
mouse embryos, and that 
35% of null mutants display pre-gastrulation and
gastrulation defects consistent with the absence or mispositioning of the AVE,
with the remainder surviving to adulthood. Our results suggest that Gdf3 has a
crucial Nodal-like activity and is likely to represent a functional homolog
for Vg1 in early mouse development.
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MATERIALS AND METHODS Cell |
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MATERIALS AND METHODS Cell
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DISCUSSION
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), and
contain a heterologous Xenopus Bmp2 prodomain and a FLAG or V5
epitope-tag inserted immediately after the proprotein convertase cleavage site
(Fig. 1A). Luciferase assays
for Gdf3 and Nodal activities were performed as described
(Yan et al., 2002).
Co-transfection of an expression construct for ß-galactosidase was used
to normalize for transfection efficiency, and addition of empty
pcDNA3 vector was used to maintain constant amounts of transfected
DNA. Protein expression was detected by western blotting using a goat
anti-mouse Gdf3 polyclonal antiserum (R&D Systems), polyclonal antiserum
against mouse Nodal (Yan et al.,
2002), polyclonal antiserum against mouse Lefty1
(Chen and Shen, 2004), or a
monoclonal anti-FLAG M2 antibody (Sigma).
Protein cross-linking and co-immunoprecipitation analysis
Reversible chemical crosslinking with 0.5 mM DTSSP
[3,3'-dithiobis(sulfosuccinimidyl proprionate)] (Pierce) and
co-immunoprecipitation were carried out as described previously
(Chen and Shen, 2004). To
detect the association of Gdf3 and Lefty1 in conditioned media, 293T cells
were transfected with pcDNA3-bf-Gdf3 and pcDNA3-Lefty1;
culture supernatants were collected 48 hours post-transfection and directly
subjected to immunoprecipitation with anti-FLAG M2 antibody (Sigma). For all
other experiments, crosslinking with DTSSP was carried out prior to
co-immunoprecipitation, using whole-cell lysates prepared with RIPA114 buffer
(Yan et al., 2002). The
kinase-inactive receptor mutants ActRIIB(K217R)-Myc and ActRIB(K234R)-HA have
been described previously (Yeo and
Whitman, 2001). Western blotting was performed using the
antibodies described above, as well as monoclonal anti-HA antibody (Covance)
and anti-Myc antibody (Santa Cruz).
Xenopus microinjection and animal cap assays
Ovulation, in vitro fertilization, and embryo and explant cultures were
carried out as described (Habas et al.,
2001; Kato et al.,
2002). Sequences corresponding to mouse Gdf3, bf-Gdf3 and Nodal
were cloned into the pCS2+ vector for in vitro transcription using a mMachine
SP6 system (Ambion). Capped mRNA was injected into the two ventral marginal
zones of the four-cell embryo for phenotypic assays or into the animal
hemisphere of Xenopus embryos at the two-cell stage. Animal caps were
explanted at blastula stages 8.5-9 and cultured to early gastrula stage 11 for
total RNA extraction and RT-PCR analysis. For phenotypic assays, embryos were
raised to stage 30 in 0.1xMMR.
Gene targeting and phenotypic analysis
Gene targeting was performed as described
(Bradley, 1987;
Matzuk et al., 1992), using a
129S6SvEv-derived ES cell line derived from AB2.1. Sixty-two percent of clones
resistant to both HAT and FIAU (n=52) were correctly targeted, as
determined by Southern blot analysis using both 5' and 3' probes.
Four independent clones were injected into mouse blastocysts, giving rise to
germline male chimeras that sired mutant mice with identical phenotypes.
Whole-mount in situ hybridization was carried out as described
(Ding et al., 1998;
Wilkinson, 1992), using probes
labeled by the DIG RNA Labeling Kit (Boehringer Mannheim). Most of the marker
analysis was performed using a C57BL/6J congenic strain, although similar data
were obtained using a C57BL/6J-129S6/SvEv mixed genetic background.
Histological analysis was performed on formalin-fixed paraffin sections of
whole decidua stained with Hematoxylin and Eosin.
Real-time RT-PCR analysis
Total RNA from individual embryos in the C57BL/6 strain background was
isolated using the RNeasy Micro Kit (Qiagen) and reverse-transcribed to
generate first-strand cDNA (Invitrogen). Real-time PCR amplification was
performed using a Mx4000 quantitative PCR instrument with SYBR Green QPCR
Master Mix (Stratagene). The primer pairs used were: for Nodal,
5' CCA TGC CTA CAT CCA GAG CCT GC 3' and 5' TGG TGT TCC AGG
AGG ACC CTG CC 3'; for Gdf1,5' TTC TGC CAG GGC ACG TGC G
3' and 5' GGA GCA GCT GCG TGC ATG AG 3'; and for
Gapdh, 5' TGC GAC TTC AAC AGC AAC TC 3' and 5' GCC
TCT CTT GCT CAG TGT CC 3'. Three independent reverse-transcription
reactions were performed on each total RNA, followed by real-time PCR
amplification in triplicate and normalization against Gapdh
levels.
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MATERIALS AND METHODS Cell
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DISCUSSION
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), and
added a FLAG or V5 epitope tag at the N terminus of the mature ligand for ease
of protein detection (Fig. 1A).
The resulting bf-Gdf3 (Bmp2 prodomain-FLAG-mature Gdf3) fusion protein is
expressed and processed efficiently in conditioned media of transfected 293T
cells (see Fig. S1A in the supplementary material). We noted that there were
several Gdf3 proprotein and mature species detected by western blotting;
consensus glycosylation sequences located in the mature Gdf3 sequence and in
the Gdf3 and Bmp2 prodomains suggest that these species differ in their levels
of N-glycosylation.
Given the body of evidence that Vg1 signals in a pathway similar to that of
Nodal, we examined the activity of these Gdf3 protein products using a
luciferase reporter assay that we developed for Nodal activity in transfected
293T cells (Yan et al., 2002).
This assay relies upon reconstitution of the essential signaling components
Cripto and Foxh1, which are not expressed in 293T cells [nor are Nodal, Gdf1,
Gdf3, Lefty1 or Lefty2 (Chen and Shen,
2004; Yan et al.,
2002) (C.C. and M.M.S., unpublished)]; moreover, Bmp4 does not
display activity in this assay (Yan et
al., 2002). When Nodal is co-transfected with Cripto and Foxh1
into 293T cells, signaling activity can be detected using the
activin/Nodal-responsive luciferase reporter A3-luc
(Fig. 1B). When co-expressed
with Cripto and Foxh1, native Gdf3 gives a low but detectable level of
activity, consistent with the low levels of mature ligand detected in
conditioned media, whereas the modified bf-Gdf3 results in significant
signaling activity (Fig. 1B;
see Fig. S1 in the supplementary material).
As Gdf3 and Nodal displayed similar activities in this cell culture assay,
we examined whether these ligands would behave identically if parameters of
this assay were altered. As is the case for Nodal
(Yan et al., 2002), we found
that Gdf3 activity was absolutely dependent upon expression of EGF-CFC
proteins and Foxh1 (Fig. 1B).
We further explored the EGF-CFC dependence of Gdf3 by examining whether other
EGF-CFC proteins could mediate Gdf3 signaling, and observed that Gdf3
displayed less activity in combination with mouse Cryptic (Cfc1) or fish Oep
than with mouse Cripto (Fig.
1C). Finally, we found that co-expression of Lefty1 or Lefty2
could inhibit Gdf3 signaling activity (Fig.
1D; data not shown), analogous to Nodal
(Chen and Shen, 2004). Taken
together, these results indicate that Gdf3 can use a signaling pathway highly
similar to that for Nodal.
Interaction of Gdf3 and Cripto in a receptor complex
To address whether Gdf3 signaling is mediated by activin receptors, and
examine the formation of a Gdf3 receptor complex, we used reversible protein
crosslinking/co-immunoprecipitation. We co-expressed epitope-tagged type I
(ActRIB/ALK4) and type II (ActRIIB) receptors together with bf-Gdf3 and
FLAG-tagged Cripto (f-Cripto) in 293T cells, and examined the proteins
immunoprecipitated together with ActRIIB following crosslinking with the
membrane-impermeable agent DTSSP (Chen and
Shen, 2004).
Interestingly, we found that bf-Gdf3 proprotein interacted equally well
with activin receptors in the absence or presence of Cripto, similar to our
previous observations with Nodal (Fig.
2A) (Chen and Shen,
2004). However, the interaction of mature Gdf3 ligand with activin
receptors was greatly enhanced in the presence of Cripto, similar to previous
observations of EGF-CFC dependent receptor complex formation with mature Nodal
in frog animal caps (Fig. 2A,
compare lanes 4 and 6) (Cheng et al.,
2003; Yeo and Whitman,
2001). We also obtained similar results with native Gdf3 in this
assay, although the levels of mature ligand present in activin receptor
complexes were low (data not shown), indicating that our findings are not a
consequence of the use of a heterologous Bmp2 prodomain. Furthermore, in
crosslinking/co-immunoprecipitation experiments in the absence of
co-transfected activin receptors, we found that Cripto interacted equally well
with the bv-Gdf3 proprotein (Bmp2 prodomain-V5-mature Gdf3) as with the mature
ligand (Fig. 2B). In
combination, these findings indicate that Cripto specifically enhances and/or
stabilizes receptor complex formation by mature Gdf3 ligand with activin
receptors.
Finally, we investigated the basis by which Lefty1 can inhibit Gdf3
signaling activity (Fig. 2C).
We and others have shown that Lefty proteins can inhibit Nodal signaling
through an interaction with EGF-CFC proteins
(Chen and Shen, 2004;
Cheng et al., 2004), as well
as with mature Nodal ligand in conditioned media
(Chen and Shen, 2004).
Consequently, we examined whether Lefty1 could interact with Gdf3 in
conditioned media in the absence of crosslinking. We found that Gdf3 could
specifically immunoprecipitate the mature Lefty1 protein, but not the Lefty1
proprotein (Fig. 2C), similar
to our previous findings with Nodal (Chen
and Shen, 2004).
Similar effects of Gdf3 and Nodal misexpression in Xenopus embryos
To examine whether Gdf3 signals in a Nodal-like pathway in vivo, we
performed gain-of-function assays in Xenopus laevis embryos to
compare the effects of Gdf3 and Nodal misexpression.
Previous studies have shown that misexpression of mouse Nodal in the
ventral marginal zone can dorsoanteriorize Xenopus embryos, and
induce dose-dependent expression of dorsal mesoderm markers in animal caps
(Jones et al., 1995;
Joseph and Melton, 1997;
Takahashi et al., 2000).
Following microinjection of mRNAs into the two ventral marginal zones of
four-cell stage embryos, we observed the formation of partial secondary axes
in response to low levels of Nodal (10 pg) and higher levels of Gdf3 or
bf-Gdf3 (100 pg and 50 pg, respectively)
(Fig. 3A-D;
Table 1). Similarly, expression
of low doses of Nodal (50 pg) or bf-Gdf3 (50 pg) in animal caps induced
morphogenetic elongation (79%, n=33 for Nodal; 39%, n=31 for
bf-Gdf3), and expression of markers of early pan-mesoderm [Xbra
(brachyury)], dorsal mesoderm [Xwnt8, chordin, Gsc
(goosecoid)] and endoderm (Mixer), but not the dorsal marker
Xnr3 (Fig. 3E-I). We
also observed morphogenetic elongation with high doses of native Gdf3 (500 pg)
(76%, n=33), as well as induction of Xbra and Mixer
(1000 pg), suggesting that unmodified Gdf3 is poorly processed in
Xenopus embryos, but possesses similar activity to bf-Gdf3
(Fig. 3I).
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). During
post-implantation development, Gdf3 expression occurs uniformly
throughout the epiblast at 5.5 and 6.0 dpc, but is not detectable in the
extra-embryonic ectoderm or visceral endoderm
(Fig. 4C-F). Notably,
Gdf3 expression appears to cease by 6.5 dpc, prior to the onset of
gastrulation, with no further expression detected by in situ hybridization
during gastrulation or through 9.5 dpc, including in primordial germ cells.
Thus, Gdf3 expression is highly restricted to the pluripotent cells
of the inner cell mass and epiblast.
Early embryonic lethality in a subset of Gdf3 homozygous null mutants
To investigate the biological functions of Gdf3, we produced mice
with a null mutation at the Gdf3 locus (see Fig. S2 in the
supplementary material). Although we could readily recover fertile and healthy
Gdf3-/- homozygous mice, we observed these were
significantly under-represented among progeny from heterozygote intercrosses
(data not shown). Consequently, we examined the phenotypes of Gdf3
mutants at embryonic stages, and found that 35% (n=127) of
Gdf3 homozygotes displayed abnormal phenotypes at 6.0 to 8.5 dpc
(Table 2). The most severely
affected embryos were either resorbing (5%), or corresponded to empty visceral
yolk sacs at 8.5 dpc (11%) (Fig.
5A). Less severely affected embryos exhibited characteristic
morphological phenotypes, including prominent constriction at the
embryonic/extra-embryonic junction, poorly differentiated anterior structures
and abnormal mesoderm derivatives (21%)
(Fig. 5B-G). Similar
frequencies of embryonic lethality, as well as embryonic phenotypes were
observed in Gdf3-/- 129SvEv inbred and C57BL/6J congenic
mice that had been backcrossed for seven generations
(Table 2; data not shown),
suggesting that the phenotypic heterogeneity is not strongly influenced by
strain background. Phenotypic analysis of embryos from homozygous by
heterozygous crosses excluded the possibility of a Gdf3 maternal
effect (C.C. and M.M.S., unpublished).
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Further analyses demonstrated severe defects in AVE formation and movement as well as loss of definitive endoderm in a subset of Gdf3 homozygotes. Using the marker Cerl (cerberus-like), we observed that definitive endoderm formation was greatly reduced or absent in 60% of (n=5) Gdf3 homozygotes at 7.0 dpc (Fig. 6G,H). Additional analyses showed that Gdf3 homozygotes had defects in expression of Hesx1 at 6.75 dpc (38%, n=8) and Hex at 6.5-7.0 dpc in the AVE (31%, n=13) (Fig. 6I-M); similar results were observed for Lhx1, which is also expressed in the AVE (Fig. 6C,D; data not shown). Notably, these analyses identified Gdf3 homozygotes with reduction or absence of the AVE (19%, n=26) (Fig. 6D,J,L), or mislocalization of the AVE in the distal visceral endoderm (8%, n=26) (Fig. 6M), suggesting a failure in AVE movement to the prospective anterior side.
Examination of less severely affected Gdf3 mutants failed to reveal additional defects in body patterning at later stages of development, including left-right axis specification (Fig. 6N,O; data not shown). In particular, morphologically normal Gdf3 homozygotes displayed correct left-sided Nodal expression in the lateral plate mesoderm at 8.0 dpc (67% four- to eight-somite stage embryos, n=9) (Fig. 6N); a few morphologically abnormal Gdf3 embryos lacked left-sided Nodal expression (Fig. 6O), but this was probably due to absence of the lateral plate mesoderm.
Surprisingly, our marker analyses also revealed an unusual subset of Gdf3 homozygotes that display a partial axis duplication. Although Gdf3 homozygotes frequently displayed reduced or absent expression of T (brachyury), which is normally found in the node and axial mesoderm at 8.0 dpc (Fig. 6P,Q), rare homozygous embryos (5%, n=57) showed twinned axial mesoderm staining, corresponding to duplicated notochords in sections (Fig. 6R,S) and indicating that secondary axis formation is an infrequent consequence of Gdf3 inactivation.
Altered expression of Nodal in Gdf3 mutant embryos
Overall, the phenotypic abnormalities observed in affected Gdf3
mutants strongly resemble those observed in mutants for several genes in the
Nodal signaling pathway (see Discussion). To assess potential effects on this
pathway, we examined expression of Nodal itself in Gdf3
homozygotes at 6.0-6.75 dpc. In wild-type embryos prior to gastrulation,
Nodal displays graded expression in the epiblast, with highest levels
in the posterior and proximal region (Fig.
6T,Z). Interestingly, we found that 2/15 (13%) Gdf3
homozygotes showed apparent downregulation of Nodal expression at 6.0
dpc, with limited staining in the proximoposterior epiblast
(Fig. 6U), whereas 3/15 (20%)
Gdf3 mutants showed strong upregulation of Nodal expression
throughout the epiblast (Fig.
6V,A'). Similarly, quantitative real-time RT-PCR analysis of
individual embryos at 6.0 dpc revealed significant downregulation of
Nodal expression in 5/26 Gdf3 homozygotes relative to
wild-type controls, while 3/26 Gdf3 homozygotes displayed significant
Nodal upregulation (see Fig. S3A in the supplementary material).
Notably, the percentage of embryos showing altered levels of Nodal
expression by real-time PCR (34%, n=73) (analysis of 26 embryos is
shown in Fig. S3A in the supplementary material; data for an additional 47 are
not shown), is similar to the percentage displaying AVE defects by marker
analyses (27%, n=26), as well as to the percentage with morphological
defects at 6.75 and 7.5 dpc (29% and 35%, respectively;
Table 2). This correlation
suggests that the Gdf3 homozygotes with AVE defects also display
altered Nodal expression at 6.0 dpc and undergo abnormal development,
while the remaining Gdf3 homozygotes are phenotypically wild
type.
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Additional marker analyses were consistent with the interpretation that
Gdf3 mutants have primary defects in AVE formation and movement, with
consequent effects on expression of Nodal. Examination of
Cripto expression at 6.75 dpc showed that most Gdf3
homozygotes showed normal expression in the posterior epiblast, with the
exception of the unusual `conical' mutants, which showed strong upregulation
throughout the epiblast (Fig.
6E',F'; data not shown). The expression patterns of
Lefty1 in the AVE at 6.0 dpc and Lefty2 in nascent mesoderm
at 6.75 dpc were similar to that described above for other AVE and mesodermal
markers, respectively (data not shown). Furthermore, expression of
Bmp4 in the extra-embryonic ectoderm is dependent upon Nodal
signaling in the adjacent proximal epiblast
(Brennan et al., 2001), but no
effects on Bmp4 expression were observed in Gdf3 mutants at
6.5 dpc (n=7) (Fig.
6G',H'), suggesting that Nodal activity in the
proximal epiblast remains at least partially intact. Finally, as Gdf1 can also
signal through a Nodal-like pathway, we examined the expression of
Gdf1, which is expressed at low levels throughout the epiblast at 5.5
dpc and becomes upregulated at 5.75 dpc
(Wall et al., 2000) (this
work). We observed a similar expression pattern and temporal onset of
Gdf1 upregulation in Gdf3 homozygotes (n=22) and
wild-type controls (n=17) at 5.5-6.0 dpc
(Fig. 6I',J'; data
not shown). In addition, quantitative real-time PCR analysis confirmed similar
levels of Gdf1 expression in individual wild-type and Gdf3
mutant embryos at 6.0 dpc (see Fig. S3B in the supplementary material).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS Cell
RESULTS
DISCUSSION
REFERENCES |
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Gdf3 functions in a Nodal-related pathway
Our studies provide three principal lines of evidence that Gdf3 acts in a
Nodal-related pathway. First, we have shown that Gdf3 signaling in cell
culture displays the defining features of the Nodal pathway in its requirement
for EGF-CFC proteins and ability to be inhibited by Lefty proteins. At the
biochemical level, Gdf3 can form complexes with activin receptors, and
interacts with both Cripto and Lefty proteins in a manner highly reminiscent
of Nodal. Second, we have demonstrated that misexpression of bf-Gdf3 and Nodal
in Xenopus embryos and animal caps results in essentially
indistinguishable phenotypes. In particular, the ability of bf-Gdf3 to induce
dorsal mesoderm markers in animal caps underscores the similarity of Gdf3 and
Nodal activities, and suggests that Gdf3 activity is specific for a Nodal-like
pathway in vivo. Finally, the majority of affected Gdf3 mutants
display phenotypes associated with loss- or reduction-of-function for
components of the Nodal signaling pathway. In particular, Nodal signaling from
the epiblast is essential for AVE formation
(Brennan et al., 2001;
Norris et al., 2002), as well
as its movement from its original distal position to the prospective anterior
side (Ding et al., 1998;
Lowe et al., 2001;
Yamamoto et al., 2004).
Failure to form the AVE leads to the absence of anterior structures and is
often correlated with an empty yolk sac phenotype
(Brennan et al., 2001;
Waldrip et al., 1998), similar
to that observed in the most severely affected Gdf3 mutants. Less
severely affected Gdf3 mutant embryos exhibit defects in mesoderm and
definitive endoderm formation, similar to Nodal null and hypomorphic
mutants (Conlon et al., 1994;
Lowe et al., 2001;
Norris et al., 2002;
Zhou et al., 1993). In
addition, Gdf3 mutants display reduced anterior structures, most
probably owing to loss of axial mesendoderm, which is commonly observed in
mutants for Nodal pathway components (Dunn
et al., 2004; Hoodless et al.,
2001; Lowe et al.,
2001; Song et al.,
1999; Vincent et al.,
2003; Yamamoto et al.,
2001).
Based on our findings, we propose that Gdf3 functions through a Nodal-like
pathway to provide a crucial signal for AVE formation and movement in
pre-gastrulation development (Fig.
7A). The variability of the Gdf3-null phenotype might
thus be due to stochastic differences in the ability of Nodal to
provide sufficient pathway activity to compensate for the absence of Gdf3.
Gdf3 may also be required subsequently for mesoderm and definitive
endoderm formation; however, as Nodal expression is perturbed in the
absence of Gdf3, we are unable to distinguish whether the
gastrulation phenotypes of Gdf3 mutants represent primary or
secondary consequences of Gdf3 inactivation. A second, non-mutually
exclusive possibility is that Gdf3 may act to upregulate Nodal
expression itself, through its potential to activate the Nodal positive
autoregulatory loop. This model is perhaps less plausible as deletion of the
Nodal autoregulatory enhancer results in a relatively mild left-right
patterning defect (Norris et al.,
2002); however, the recent elucidation of a second
Nodal-responsive enhancer raises the possibility that autoregulation may play
an important role at earlier stages of development
(Saijoh et al., 2005).
Unexpectedly, we have observed that Nodal expression can either be
downregulated or upregulated in the absence of Gdf3. These apparently
opposite outcomes can potentially be explained by the requirement of
Gdf3 for AVE induction and movement. In the absence of Gdf3,
the AVE may be able to form, but be unable to move, leading to retention of
Nodal expression in the proximal epiblast, resulting in reduction or
absence of mesoderm and definitive endoderm formation
(Fig. 7B). However, if the AVE
fails to form, Nodal expression would remain at high levels
throughout the epiblast (Fig.
7B), similar to the phenotype of Smad2-null mutants
(Brennan et al., 2001;
Waldrip et al., 1998). In
addition, overexpression of Nodal might lead to expansion and
splitting of the primitive streak at early gastrulation stages, as well as
partial axis duplications, as expression of Nodal pathway inhibitors in the
AVE is necessary to suppress formation of a secondary axis
(Bertocchini and Stern, 2002;
Perea-Gomez et al., 2002).
Similar phenotypes have been observed in other cases of Nodal pathway
upregulation, such as in mutants for the transcriptional repressor
Drap1 (Iratni et al.,
2002), or combined inactivation of the Nodal antagonists
Lefty1 and Cerl
(Perea-Gomez et al.,
2002).
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), as
well as for the Xenopus Tgfß factor Derriere
(Eimon and Harland, 2002).
Whether such heterodimerization is physiologically relevant remains to be
elucidated.
|
),
and has been proposed to cooperate with Wnt and FGF signals in primitive
streak formation and initiation of Nodal expression
(Bertocchini et al., 2004;
Skromne and Stern, 2001).
Similarly, Xenopus Vg1 transcripts are localized to vegetal cells at
the blastula stage, and have been suggested to play a central role in dorsal
mesoderm and endoderm formation (Joseph
and Melton, 1998; Kessler and
Melton, 1995; Thomsen and
Melton, 1993; Weeks and
Melton, 1987). These putative functions of Vg1-related
genes are generally consistent with our findings that mouse Gdf3
mutants display defects in formation of the AVE, mesoderm and definitive
endoderm.
Interestingly, the conserved functions of Xenopus Vg1 may have
been divided between Gdf1 and Gdf3, its mammalian
counterparts. The patterns of Gdf1 and Gdf3 expression in
the epiblast are strikingly complementary, with Gdf3 being
downregulated shortly after 6.0 dpc and Gdf1 being upregulated at
5.75 dpc, when it can be detected throughout the epiblast
(Wall et al., 2000) (this
work). Null mutants for Gdf1 are defective for left-right axis
specification (Rankin et al.,
2000), consistent with the proposed role for Vg1 in
coordinating left-right patterning in Xenopus
(Hyatt et al., 1996;
Hyatt and Yost, 1998;
Kramer and Yost, 2002).
Notably, the phenotype of Gdf1 mutants is indistinguishable from that
of Cryptic mutants (Yan et al.,
1999), suggesting that Gdf1 signaling may be facilitated by
Cryptic in vivo.
The potential overlapping functions of Nodal, Gdf3 and Gdf1 suggests that
the Nodal signaling pathway may play as yet poorly understood roles in
pre-implantation and early post-implantation development. This possibility is
supported by the expression patterns of Gdf3 and Nodal in
pre-gastrulation embryos, as well as by the conserved expression of Nodal,
Cripto, Lefty1 and Gdf3 in undifferentiated mouse and human ES
cells (Besser, 2004;
Bhattacharya et al., 2004;
Ramalho-Santos et al., 2002;
Sato et al., 2003). Moreover,
recent studies have also suggested a role for Nodal signaling in maintaining
the undifferentiated state of human ES cells
(James et al., 2005;
Vallier et al., 2004). These
observations lead us to speculate that this pathway may play a broader role in
mammalian embryogenesis than previously conceived.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/2/319/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS Cell
RESULTS
DISCUSSION
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D. Mesnard, M. Guzman-Ayala, and D. B. Constam Nodal specifies embryonic visceral endoderm and sustains pluripotent cells in the epiblast before overt axial patterning Development, July 1, 2006; 133(13): 2497 - 2505. [Abstract] [Full Text] [PDF]
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-Nodal antibody or 
