1 Center for Advanced Biotechnology and Medicine and Departments of Pediatrics,
UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
2 Centre for Developmental and Biomedical Genetics, Department of Biomedical
Science, University of Sheffield, Sheffield S10 2TN, UK
Author for correspondence (e-mail:
mshen{at}cabm.rutgers.edu)
Accepted 11 October 2005
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SUMMARY |
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Key words: EGF-CFC proteins, Axial mesendoderm, Chimera analysis, Non-cell-autonomy, Co-receptor, Nodal signaling, Mouse
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Introduction |
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Molecular genetic studies in several vertebrate systems have identified
EGF-CFC proteins as essential components of the Nodal signaling pathway, and
have demonstrated their ability to act in cis as co-receptors for Nodal
ligands (Gritsman et al.,
1999; Kumar et al.,
2001
; Reissmann et al.,
2001
; Yan et al.,
2002
; Yeo and Whitman,
2001
). EGF-CFC proteins are small extracellular proteins that
contain a divergent epidermal growth factor (EGF) motif and a novel conserved
cysteine-rich domain (termed the CFC motif) (reviewed by
Shen and Schier, 2000
), and
they are attached to the cell membrane through a glycosyl-phosphatidylinositol
(GPI) linkage (Minchiotti et al.,
2000
). Two EGF-CFC genes, Cripto
(Tdgf1) and Cryptic (Cfc1 - Mouse Genome
Informatics), have been identified in mammals; other family members include
chick Cripto, frog FRL-1 and zebrafish one-eyed
pinhead (oep). In the case of oep, cell transplantation
experiments have shown its cell-autonomy in prechordal plate, floor plate, and
endoderm (Gritsman et al.,
1999
; Schier et al.,
1997
; Strahle et al.,
1997
). These data, together with the absence of phenotypic
consequences following oep overexpression in zebrafish embryos
(Zhang et al., 1998
), are
consistent with a strict role for Oep as a cis-acting co-receptor for
Nodal.
Zygotic oep mutants lose axial mesendoderm and display associated
ventral midline and forebrain defects
(Schier et al., 1997). Despite
a relatively low overall level of sequence conservation, all EGF-CFC
family members appear to have functionally similar activities in assays for
phenotypic rescue of oep mutants
(Zhang et al., 1998
). However,
it has been unclear to date whether, like Oep, mammalian EGF-CFC proteins are
required in axial midline formation and forebrain patterning, and whether they
function as cis-acting co-receptors for Nodal in this context. Intriguingly,
by contrast with Oep, studies of Cripto function have raised the possibility
that it can function as a secreted trans-acting signaling factor
(Bianco et al., 2003
;
Kannan et al., 1997
;
Minchiotti et al., 2001
;
Parisi et al., 2003
;
Yan et al., 2002
).
Furthermore, a previous low-resolution chimera analysis has suggested
potential non-cell-autonomy for Cripto
(Xu et al., 1999
), although
the absence of cell-marking in this experiment precluded analysis of specific
requirements for Cripto function during embryogenesis.
In the studies described below, we show that Cripto, like other Nodal pathway components, is required for axial mesendoderm and definitive endoderm formation in mouse embryogenesis. However, we find that this requirement for Cripto is non-cell-autonomous, and provide evidence that Cripto can act as a soluble trans-acting factor in vivo. Complementary gain-of-function approaches in chick embryos reveal effects of exogenous soluble Cripto protein on differentiation of chick node/head process mesoderm cells, suppressing posterior axial mesoderm fates and promoting anterior mesendodermal fates. Our findings potentially resolve long-standing discrepancies concerning the mechanisms of Cripto function, and suggest that a GPI-linked protein can act as a trans-acting signal.
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Materials and methods |
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We used the EIIa-Cre transgenic line to obtain both partial and
complete deletions in vivo, taking advantage of the low Cre activity in the
germline of EIIa-Cre males (Xu et
al., 2001). We obtained all three possible deletion products with
approximately equal frequency in the progeny of C57BL/6 wild-type females
x EIIa-Cre; Cripto3loxP/+ males, including the
desired Criptodel and Criptoflox
alleles. (Formal allele names are as follows:
CriptolacZ=Criptotm1Mms; Cripto3loxP=
Criptotm2Mms;
Criptodel=Criptotm2.1Mms;Criptoflox=Criptotm2.2Mms).
PCR primers used for genotyping were: Cripto3loxP
(forward) 5'-CCT CCC AAG TTC ACT ACC AAA TCT-3', (reverse)
5'-TCC CCA CCA TCC ACC ACC AAG TAG-3';
Criptodel (forward) 5'-AGC CAT CTC ACC AGC CTT
CA-3', (reverse) 5'-CAT CTG GGA CAT GCC CAC TA-3';
CriptolacZ (forward) 5'-CCA TCC CCT GCC CGT CTA CAC
G-3', (reverse) 5'-GTC ACG CAA CTC GCC GCA CAT-3'.
Histology, in situ hybridization, and ß-galactosidase staining
Whole-mount in situ hybridization and ß-galactosidase staining were
performed as described (Ding et al.,
1998). Genotypes of 6.5 days-post-coitum (dpc) embryos were
determined by PCR using genomic DNA from cultured extraplacental cone
(Ang and Rossant, 1994
); for
7.5 dpc embryos and older, extra-embryonic tissues following in situ
hybridization were lysed for DNA extraction and PCR. Following whole-mount in
situ hybridization, embryos were embedded in OCT and 6 µm cryosections were
counterstained with Methyl Green, or left unstained. Histological analysis was
performed by hematoxylin and eosin staining of paraffin sections of embryos
fixed in 10% formalin. Mouse probes for in situ hybridization were:
Cer1 (Belo et al.,
1997
); Cripto (Ding
et al., 1998
); En1
(Davis and Joyner, 1988
);
Fgf8 (Crossley and Martin,
1995
); Foxa2 (Ang and
Rossant, 1994
); Gsc
(Belo et al., 1997
);
Hex (Hhex) (Thomas et
al., 1998
); Shh
(Echelard et al., 1993
); and
Six3 (Oliver et al.,
1995
). Chick probes were Gsc
(Hume and Dodd, 1993
) and
Hex (Crompton et al.,
1992
).
Chimera analysis
Chimeras were generated by morula aggregation
(Nagy et al., 2003), using 4-8
cell embryos from pregnant C57BL/6 wild-type mice and from a
Rosa26/Rosa26; Criptonull/+ x
CriptolacZ/+ cross (here we refer to the
Gt(ROSA)26Sor gene-trap allele as Rosa26). Embryos were
recovered at 7.5-9.5 dpc, and genotypes determined by PCR analysis of DNA
extracted from extra-embryonic tissues. Whole-mount embryos were stained for
ß-galactosidase activity, and cryosections were counterstained with
Nuclear Fast Red (Vector Laboratories). In a second method of chimera
analysis, marked ES cell lines were established from blastocysts obtained by a
Rosa26/Rosa26; Criptodel/+ x
CriptolacZ/+ cross, using standard methods
(Nagy et al., 2003
). The
resulting ES colonies were genotyped by PCR, and a Rosa26/+;
CriptolacZ/del cell line and a Rosa26/+;
CriptolacZ/+ line were established. Chimeras were generated by
injecting approximately 5-10 ES cells into C57BL/6 blastocysts.
Chick explant cultures, in-ovo operations and immunofluorescence
Embryos and explants were assayed according to standard techniques
(Placzek et al., 1993;
Placzek et al., 1990
). For
explant culture experiments, posterior head process mesoderm or Hensen's node
explants were grown for 20 hours in Optimem (Invitrogen) containing 2% fetal
calf serum, 1% penicillin/streptomycin, and 1% L-glutamine, either alone or
with addition of 10-fold concentrated 293T culture supernatants. 293T cells
were either untransfected or transfected with full-length mouse
Cripto, which results in production of active Cripto protein in
conditioned medium (Minchiotti et al.,
2000
; Yan et al.,
2002
). The small molecule inhibitor SB-431542 was used as
described previously (Inman et al.,
2002
). For in-ovo experiments, beads were grafted at
Hamburger-Hamilton (HH) stage 3-4 and embryos developed until HH stage 8-9.
Explants and embryos were fixed in 4% paraformaldehyde for 2 hours and
embedded in OCT for cryosectioning. Sections were processed for
immunofluorescence detection using the 3B9 monoclonal antibody [1:10 dilution
from culture supernatant (Placzek et al.,
1990
)] or the Shh monoclonal antibody
(Ericson et al., 1996
) and a
Cy3-labeled anti-mouse secondary antibody (Jackson ImmunoResearch; 1:200
dilution), as described previously
(Placzek et al., 1993
).
Quantitative RT-PCR
Real-time quantitative RT-PCR was performed using the ABI Prism 7700
sequence detection system (Applied Biosystems). RNA was standardized using
ß-actin amplification as an internal control. Primers used were:
Gsc (forward) 5'-AAA AGA CGG CAC CGG ACT ATC-3',
(reverse) 5'-TCG TTT CCT GGA AGA GGT TTT C-3', and the probe
5'-CAC TGA CGA GCA GCT CGA AGC GC-3' (labeled with the reporter
dye FAM on the 5' nucleotide and the quenching dye TAMRA on the 3'
nucleotide). Fgf10 (forward) 5'-ATG ACC TCG GCC AGG ACA
T-3', (reverse) 5'-TGA TTG TAG CTC CGC ACG TG-3', and probe
5'-CTG TCC CCG GAG GCC ACC AA-3' labeled with the reporter Yakima
Yellow on the 5' nucleotide and the quenching dye Eclipse Dark Quencher
on the 3' nucleotide. ß-actin (forward) 5'-GGT CAT CAC CAT
TGG CAA TG-3', (reverse) 5'-CCC AAG AAA GAT GGC TGG AA-3'
and the fluorogenic probe 5'-TTC AGG TGC CCC GAG GCC CT-3' labeled
with the reporter dye Yakima Yellow on the 5' nucleotide and the
quenching dye Eclipse Dark Quencher on the 3' nucleotide. Probes were
supplied by Eurogentec. Relative quantification of Gsc and
Fgf10 mRNA was calculated using the standard curve method.
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Results |
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To determine the cell types that derive from this transient expression of
Cripto, we took advantage of the CriptolacZ null
allele, in which the lacZ reporter gene is under the control of the
Cripto promoter (Ding et al.,
1998). This knock-in allele recapitulates the pattern of
endogenous Cripto expression, except for a temporal delay that
probably corresponds to the perdurance of ß-galactosidase
(Ding et al., 1998
). In
CriptolacZ/+ heterozygotes at late neural plate stages, we
found ß-galactosidase staining throughout the mesoderm and definitive
endoderm, with strongest staining in the axial mesendoderm
(Fig. 1D,E). Taken together,
these data indicate that Cripto-expressing cells give rise to axial
mesendoderm and anterior definitive endoderm.
Generation of a hypomorphic allele of Cripto
Cripto null mutants display early embryonic lethality due to a
failure in anterior-posterior axis positioning before gastrulation
(Ding et al., 1998).
Therefore, to investigate subsequent functions of Cripto in
embryogenesis, we have generated novel hypomorphic (partial loss-of-function),
conditional, and null alleles for Cripto
(Fig. 1F-H). In our targeting
strategy, a `3-loxP' construct containing a `floxed' (flanked by
loxP sites) PGK-neo selection cassette was inserted at the
Cripto locus. Following germline transmission of this allele, mice
carrying this Cripto3loxP allele were crossed with
Ella-Cre transgenic mice to produce all three possible derivatives
from Cre-loxP mediated recombination (Xu
et al., 2001
). This strategy resulted in the generation of a new
null allele corresponding to the deletion of exons 3, 4 and 5
(Criptodel) (Fig.
1F), as well as a conditional floxed allele
(Criptoflox) (data not shown). Both homozygous
Criptodel and heteroallelic
CriptolacZ/del embryos displayed a phenotype identical to
that of CriptolacZ homozygotes (data not shown).
As the intronic insertion of selection cassettes with strong transcription
termination sequences frequently leads to the generation of a hypomorphic
allele (Meyers et al., 1998),
we performed genetic crosses to determine whether the
Cripto3loxP allele might be hypomorphic. Although
homozygous Cripto3loxP/Cripto3loxP mice can be
recovered as adults at Mendelian ratios
(Table 1A), we observed that
progeny from crosses of Cripto3loxP/+ and
CriptolacZ/+ mice were significantly under-represented for
the Cripto3loxP/lacZ genotype
(Table 1B). The surviving mice
with this genotype can reach adulthood and breed successfully; gross and
histological examination of these mice has not yet revealed any phenotypic
abnormality (J.C. and M.M.S., unpublished). Because
Cripto3loxP/del embryos displayed an identical phenotype
to Cripto3loxP/lacZ embryos (data not shown), we refer to
both Cripto3loxP/lacZ and
Cripto3loxP/del embryos as having a
Cripto3loxP/null genotype.
|
Next, we examined the expression of specific tissue markers in severely
affected Cripto3loxP/null embryos to ascertain the basis
for these phenotypic defects. Sonic hedgehog (Shh) is
expressed in ventral midline structures and gut endoderm at 8.25 dpc, but was
often greatly reduced or absent in Cripto3loxP/null
embryos (71%, n=7) (Fig.
3A). Similarly, Hex is a marker of anterior definitive
endoderm at 7.5 dpc, but was often undetectable in
Cripto3loxP/null mutants (40%, n=15)
(Fig. 3B); by contrast,
Hex expression in the anterior visceral endoderm at 6.5 dpc was
unaffected (n=5; data not shown). Analyses of neural markers revealed
that severely affected Cripto3loxP/null embryos retain
expression of the midbrain-hindbrain marker En1 (n=4)
(Fig. 3C). However,
Cripto3loxP/null embryos often displayed truncation of the
rostral forebrain, as shown by loss of anterior expression for Six3
(67%, n=9) and Fgf8 (63%, n=8)
(Fig. 3D-F). The loss of
Hex expression, in particular, suggests that the forebrain defects
may represent a secondary consequence of the loss of axial mesendoderm
derivatives, notably the anterior definitive endoderm and prechordal plate
(reviewed by Kiecker and Niehrs,
2001; Wilson and Houart,
2004
).
Consistent with this interpretation, histological and marker analyses of Cripto3loxP/null embryos during gastrulation stages demonstrated severe defects in the formation of axial mesendoderm and definitive endoderm. In wild-type embryos, the prechordal plate is evident from the apposition of axial mesendoderm to anterior ectoderm at the midline (Fig. 2L), whereas an intact prechordal plate did not form in Cripto3loxP/null mutants (Fig. 2M,N). At head-fold stages, Shh is expressed in the node, notochordal plate and prechordal plate, but in Cripto3loxP/null mutants was either absent or occasionally observed in scattered patches of mesodermal cells that were displaced from the midline (53%, n=19) (Fig. 4A-D). Similar results were obtained with the axial midline markers Foxa2 (HNF3ß) (36%, n=14) (Fig. 4E-H) and chordin (Chrd, 67%, n=10/15) (data not shown). Expression of goosecoid (Gsc) marks prechordal plate, adjacent midline neuroectoderm and foregut endoderm, but was abolished in approximately half of Cripto3loxP/null mutants (50%, n=6) (Fig. 4I-L). Finally, expression of cerberus 1 homolog (Cer1) showed the nearly complete loss of definitive endoderm in strongly affected Cripto3loxP/null embryos (50%, n=4) (Fig. 4M-P), consistent with the results with Hex (Fig. 3B).
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Addition of soluble Cripto to chick tissues suppresses posterior mesodermal fates and promotes anterior mesendodermal fates
The non-autonomous function of Cripto suggests that Cripto might
act as a secreted trans-acting factor in the differentiation of anterior
mesendoderm cell types, a process known to be dosage-sensitive for Nodal
pathway activity (Agius et al.,
2000; Dunn et al.,
2004
; Gritsman et al.,
2000
; Vincent et al.,
2003
). Consistent with this possibility, chick Cripto
(also known as CFC) is expressed in the anterior primitive
streak/Hensen's node at HH stages 3 and 4, as well as in notochord and
prechordal plate at stages 5 and 6 (Colas
and Schoenwolf, 2000
; Schlange
et al., 2001
). To determine whether soluble Cripto protein might
affect the differentiation of anterior mesendodermal cell types, we used chick
embryos to assay the activity of Cripto-containing conditioned medium
(Minchiotti et al., 2000
;
Yan et al., 2002
).
Initially, we examined cell differentiation in node or head process mesoderm explants cultured in control medium, or medium conditioned by untransfected 293T cells. When HH stage 4--4 Hensen's node explants were cultured under these conditions (Fig. 7A), Gsc-expressing prechordal mesoderm cells, Hex-expressing anterior endoderm cells and 3B9-expressing notochord cells differentiated from the explanted node (n=20; Fig. 7B-D, data not shown). By contrast, when posterior head process mesoderm from HH stage 5 embryos was cultured (Fig. 7E), neither Gsc-expressing prechordal mesoderm cells nor Hex-expressing anterior endoderm cells was detected; however, differentiating notochord cells marked by the cell surface marker 3B9 were detected (n=30) (Fig. 7F-H, data not shown).
By contrast, addition of Cripto-conditioned medium evoked a very distinct pattern of differentiated cell types within node and head process mesoderm explants. When Cripto-conditioned medium was added to HH stage 4--4 Hensen's node explants, the differentiation of 3B9-expressing notochord cells was prevented (80%, n=20) (Fig. 7L), but robust differentiation of Gsc- and Hex-expressing cells was observed (100%, n=16) (Fig. 7J,K). This suggests that soluble Cripto protein can promote the differentiation of more anterior mesendodermal cell types (prechordal mesoderm and anterior endoderm), at the expense of posterior notochord cells. In support of this interpretation, when HH stage 5 posterior head process mesoderm was exposed to Cripto, Gsc-expressing prechordal mesoderm cells were detected in the central component of the explant, while Hex-expressing anterior endodermal cells were detected in peripheral regions of the explant (80%, n=10) (Fig. 7N,O). Concomitantly, the differentiation of 3B9-expressing notochord cells was prevented (80%, n=10) (Fig. 7P).
|
We next established whether Cripto could similarly elicit a change in fate of axial mesodermal cells in vivo, promoting a prechordal mesoderm fate at the expense of notochord cell fate. Beads soaked in medium containing Cripto protein, or control medium, were implanted adjacent to Hensen's node of HH stage 4--4 chick embryos (n=6 each; Fig. 7Q), and the embryos were allowed to develop in ovo to HH stage 8-9 (Fig. 7U). In control-operated embryos, axial mesoderm displayed the normal phenotypic properties of notochord cells, as no expression of Gsc was detected (Fig. 7R), but the cells co-expressed Shh and 3B9 (Fig. 7S,T). By contrast, in embryos exposed to Cripto-conditioned medium, axial mesoderm at an equivalent rostrocaudal level (Fig. 7U) appeared to be anteriorized. Although situated adjacent to somites, axial mesoderm cells expressed the prechordal mesoderm marker, Gsc (Fig. 7V), as well as Shh (Fig. 7W), but expression of 3B9 was eliminated (Fig. 7X).
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Discussion |
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Role of Cripto in axial midline formation
The requirement of Cripto in axial mesendoderm and definitive
endoderm strongly indicates that Cripto is required for Nodal signaling in
these tissues and/or their progenitors. Indeed, null and/or hypomorphic mouse
mutants for several Nodal pathway components display similar phenotypes to
those of Cripto hypomorphs, including mutants for Nodal
(Lowe et al., 2001;
Norris et al., 2002
), the type
II activin receptors ActRIIA and ActRIIB
(Song et al., 1999
), the
intracellular signal transducers Smad2 and Smad3
(Dunn et al., 2004
;
Heyer et al., 1999
;
Vincent et al., 2003
), Smad4
(Chu et al., 2004
), and the
winged-helix transcription factor FoxH1
(Hoodless et al., 2001
;
Pogoda et al., 2000
;
Sirotkin et al., 2000
;
Yamamoto et al., 2001
).
Nonetheless, we note that Cripto may also have Nodal-independent functions.
For example, the EGF-CFC protein Oep has been proposed to act independently of
Nodal in regulation of cell motility in the zebrafish blastoderm
(Warga and Kane, 2003
). In
addition, cell culture studies have shown that Cripto can block Activin
signaling in the absence of Nodal activity
(Adkins et al., 2003
;
Gray et al., 2003
). Finally, a
recent study has found that the Xenopus EGF-CFC protein FRL-1 can
mediate signaling by Wnt11 in the canonical Wnt/ß-catenin pathway during
dorsal axis formation (Tao et al.,
2005
).
|
Our analyses of mutant mice show that reductions of Cripto
activity affect rostral midline structures (prechordal plate) in preference to
caudal structures (notochord), as indicated by the spectrum of
holoprosencephaly phenotypes in Fig.
2, while exposure of chick cells to exogenous Cripto both in vitro
and in vivo increases prechordal mesoderm at the expense of notochord. These
results complement previous observations that levels of Nodal
activity specify the rostrocaudal identity of axial mesoderm
(Agius et al., 2000;
Gritsman et al., 2000
;
Lowe et al., 2001
;
Norris and Robertson, 1999
;
Thisse et al., 2000
;
Vincent et al., 2003
). More
generally, our findings demonstrate that reduction of Cripto activity
can lead to a `pinhead' phenotype that resembles that of zygotic oep
mutants (Schier et al., 1997
).
This observation, together with the identification of a Cripto
loss-of-function mutation in a human patient with midline forebrain defects
(de la Cruz et al., 2002
),
provides additional support for the evolutionary conservation of
EGF-CFC functions.
Non-cell-autonomy of Cripto implies its intercellular activity
In principle, the experimental observation of non-cell-autonomy implies
that the gene product, or one of its major regulatory targets, mediates a
cell-cell signaling event. In some cases, genes encoding proteins with
intracellular functions, such as transcription factors, can nonetheless
display non-cell-autonomy (e.g. Rhinn et
al., 1999). However, analysis of the Nodal pathway component FoxH1
has shown that this transcription factor is required cell-autonomously for
axial mesendoderm and definitive endoderm formation
(Hoodless et al., 2001
). Thus,
the upstream extracellular component Cripto acts non-cell-autonomously in the
Nodal pathway, but the downstream transcription factor FoxH1 is
cell-autonomous (Fig. 8A).
Moreover, chimera analysis of Smad2 has demonstrated its
cell-autonomous requirement in definitive endoderm
(Tremblay et al., 2000
). Based
on this pathway relationship (Fig.
8A), we conclude that it is Cripto itself, not one of its
regulatory targets, that can act in trans as an intercellular mediator of
Nodal signaling.
The intercellular activity of Cripto may reflect its ability to be released
from the cell surface in an active form, as we have previously shown
(Yan et al., 2002). As Cripto
is a GPI-linked protein, several possible mechanisms may explain its release
from the cell surface. These include protein shedding due to cleavage of the
GPI anchor (e.g. Kondoh et al.,
2005
; Metz et al.,
1994
), or formation of membrane vesicles (argosomes) that can
traverse neighboring cells (Greco et al.,
2001
). Moreover, there are several precedents for shedding of
GPI-linked proteins under physiological circumstances, such as the regulated
proteolytic cleavage of ephrin-A2 during neuronal axon repulsion
(Hattori et al., 2000
), or the
release of the glypican Dally-like, which modulates Wingless signaling in
Drosophila (Kreuger et al.,
2004
). However, we note that our findings show only that Cripto is
capable of acting intercellularly under physiological circumstances in vivo,
but do not necessarily imply that such an activity is utilized during
wild-type development. For example, GFR
is a GPI-linked co-receptor
that has been proposed to act in trans to provide a response to GDNF family
ligands in cells that express the Ret receptor but not GFR
(e.g.
Mikaels-Edman et al., 2003
;
Paratcha et al., 2001
), but
this model has been challenged by a recent study showing the absence of
Ret-independent GFR
function in vivo
(Enomoto et al., 2004
).
Our data may provide a unifying basis for two bodies of literature on
Cripto activity and function that have previously seemed difficult to
reconcile. While work in zebrafish, frog and mouse systems has supported a
role for EGF-CFC proteins as cis-acting co-receptors for Nodal
ligands, there is also ample evidence for trans-acting soluble
activities of Cripto, particularly in cell culture. Notably, work from the
Salomon laboratory has shown that recombinant Cripto protein or peptide
fragments can elicit specific responses in cell culture, for example resulting
in activation of the MAPK pathway (Bianco
et al., 2002; Bianco et al.,
1999
; Bianco et al.,
2003
; De Santis et al.,
1997
; Ebert et al.,
1999
; Kannan et al.,
1997
). In some cases, these cellular responses are due to
activation of the Nodal pathway, whereas other Cripto activities appear to be
Nodal-independent (Bianco et al.,
2002
; Bianco et al.,
2003
). Other investigators have found that truncated Cripto
protein lacking the C-terminal GPI anchor can rescue the cardiac
differentiation defects of Cripto-deficient ES cells
(Parisi et al., 2003
), and
that injection of truncated Oep protein into the syncytial yolk layer of
zebrafish embryos will rescue the oep mutant phenotype
(Minchiotti et al., 2001
).
Finally, our studies using a reconstituted Nodal signaling assay in mammalian
cells have shown that the ability of soluble Cripto to signal in this system
is Nodal- and FoxH1-dependent (Yan et al.,
2002
). All of these data support a model in which Cripto possesses
intrinsic activity as a non-membrane-associated protein that can mediate Nodal
signaling in trans (Fig.
8B,C).
Consistent with possible trans-acting functions, several studies
have reported phenotypes associated with overexpression/ectopic expression of
EGF-CFC genes in vivo. For example, overexpression of wild-type human
Cryptic - but not mouse Cryptic - in zebrafish leads to
developmental delays and abnormal gastrulation movements
(Bamford et al., 2000), while
overexpression of secreted forms of oep can promote dorsoanterior
development (Kiecker et al.,
2000
). In chick embryos, implantation of cells expressing human
Cryptic, mouse Cryptic or chick Cripto on the right
side of Hensen's node leads to randomization of cardiac looping
(Schlange et al., 2001
).
Finally, overexpression of frog FRL-1 in Xenopus animal caps
results in neural induction in the absence of mesoderm formation
(Kinoshita et al., 1995
;
Yabe et al., 2003
;
Yokota et al., 2003
).
By contrast, other studies have shown the absence of a gain-of-function
phenotype following overexpression of wild-type oep in fish
(Bamford et al., 2000;
Gritsman et al., 1999
;
Zhang et al., 1998
). Moreover,
rigorous cell transplantation assays have established the strict cell-autonomy
of oep function in several tissues
(Gritsman et al., 2000
;
Schier et al., 1997
;
Strahle et al., 1997
;
Warga and Kane, 2003
). These
opposite findings in zebrafish versus mice (and chick) strongly suggest an
underlying divergence in the mechanism of EGF-CFC function. Therefore, despite
the evolutionary conservation of EGF-CFC function in axial midline
formation, EGF-CFC proteins may utilize disparate non-conserved molecular
mechanisms for this essential developmental process.
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ACKNOWLEDGMENTS |
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Footnotes |
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