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First published online 8 December 2005
doi: 10.1242/dev.02188
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1 Department of Developmental Biology, Harvard School of Dental Medicine, 188
Longwood Avenue, Boston, MA 02115, USA.
2 Department of Life Sciences and Center for Cell Signaling Research, Ewha
Women's University, Seoul 120-750, Korea.
* Author for correspondence (e-mail: mwhitman{at}hms.harvard.edu)
Accepted 27 October 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: EGF-CFC factor, Nodal, Left-right patterning, Cripto, XCR, Xenopus
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Ciccodicola et al., 1989;
Dono et al., 1991;
Dono et al., 1993;
Shen et al., 1997),
FRL1 in frogs (Kinoshita et al.,
1995), one-eyed pinhead, oep, in zebrafish
(Zhang et al., 1998) and
Cripto/CFC in chick (Colas and
Schoenwolf, 2000; Schlange et
al., 2001). EGF-CFC family members share two conserved domains: an
EGF-like domain and a CFC domain, which are conserved only among EGF-CFC
family members (reviewed by Adamson et al.,
2002; Minchiotti et al.,
2002; Saloman et al.,
2000; Shen and Schier,
2000). Recent genetic and biochemical studies have demonstrated
that EGF-CFC proteins act as essential co-factors for signaling by the
nodal/GDF1 subset of TGFß superfamily ligands (reviewed by
Saloman et al., 2000;
Schier, 2003). EGF-CFC
proteins facilitate the association of nodal and GDF1/VG1 ligands with Type I
and Type II transmembrane receptors (Cheng
et al., 2003; Reissmann et
al., 2001; Yan et al.,
2002; Yeo and Whitman,
2001). The EGF-like domain mediates association with nodal
ligands, whereas the CFC domain interacts with the receptor complex.
Loss-of-function studies have established that EGF-CFC proteins function as
co-factors for nodal/GDF1 signals in both mesendoderm and left-right axis
specification (Bamford et al.,
2000; Ding et al.,
1998; Gaio et al.,
1999; Gritsman et al.,
1999; Linask et al.,
2003; Schlange et al.,
2001; Xu et al.,
1999; Yan et al.,
1999).
The only reported frog EGF-CFC family member, FRL1, was originally
identified in a screen for FGF receptor ligands
(Kinoshita et al., 1995).
Ectopic expression and antisense oligonucleotide studies have indicated that
FRL1 is essential for the activation of ERK and subsequently for
neural formation (Yabe et al.,
2003), and is also an essential co-factor for the dorsal
determinant WNT11 (Tao et al.,
2005). Strikingly, however, no defects in either the transduction
of nodal/GDF1 signals or left-right patterning were observed following
FRL1 depletion, suggesting that the function of FRL1 might differ
from that of EGF-CFC factors in other vertebrate model organisms. We report
here the identification of two additional EGF-CFC family members in
Xenopus. The three frog EGF-CFC factors show distinct spatial and
temporal expression patterns during development. We have examined the effects
of both loss and gain of function of one of these factors, XCR2, and
have found that it is specifically required for normal left-right patterning
of XNR1 and XATV expression, and for visceral asymmetry.
XCR2 is expressed bilaterally at neurula-tailbud stages, but is
required only on the left side for normal patterning. The requirement of
XCR2 for left-side-specific expression of the nodal-related gene
XNR1, is consistent with prior work demonstrating that the expression
of nodal-related ligands are maintained by a FAST1/FOXH1-mediated
positive-feedback loop (Adachi et al.,
1999; Norris et al.,
2002; Osada et al.,
2000; Saijoh et al.,
2000). In addition, we found that ectopic expression of
XCR2 on the right side is sufficient to reverse left-right polarity,
and this effect requires XCR2 activity as a co-receptor for
nodal/GDF1 ligands. These observations indicate not only that XCR2 is
required for nodal signaling during left-right patterning, but also that
limitation of nodal signaling across the left-right axis by XCR2 can determine
the polarity of the nodal gradient across this axis.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), we
identified EST clones corresponding to two novel Xenopus laevis
EGF-CFC factors: (1) from the NIBB Mochii normalized Xenopus laevis
neurula library, cDNA clone XL044d20 (GenBank UniGene Xl. 15503); and (2) from
the NICHD_XGC_Emb1 Xenopus laevis library, cDNA clone IMAGE
Consortium CloneID 6864330 (GenBank CA987644). We designated these novel
EGF-CFC factors XCR2 and XCR3S (short form),
respectively.
To generate pCS-XCR2, pCS2-XCR3S, pCS2-hCFC1, pCRII-XNR1 and pCRII-XATV,
the ORF region of each EST clone (XCR2 or XCR3S) or cDNA insert of
pSP64PA-humanCFC1 (Bamford et al.,
2000), pCS2+XNR1 (Lustig et
al., 1996) or pCS2+XATV
(Cheng et al., 2000) was
subcloned into pCS4+, a derivative of pCS2+ described by Yeo and Whitman
(Yeo and Whitman, 2001), into
the pCS2+ vector or into the pRII vector (Invitrogen). To generate pCS2-XCR3L
(long form), the ORF region of XCR3L was amplified by PCR from cDNA of stage
10.5 embryos, and subcloned into the pCS2+ vector. pCS-XCR2 mEGF (mutated EGF)
and pCS-XCR2 mCFC (mutated CFC) were generated by PCR-based subcloning: Asn93
and Thr96 to Gly93 and Ala96 in XCR2 mEGF1, and His129 and Trp132 to Gly129
and Gly132 in XCR2 mCFC. All constructs generated by PCR amplification were
sequence verified.
Xenopus laevis genomic clones of XCR2 were isolated by PCR using the following primers: up (5'-AAGCAATTTCACATCAAC-3') and down (5'-GGTGGGCCCCGCTGCCTCTAATG-3'; the linker sequence is underlined). A splice-site-targeted antisense morpholino oligonucleotide, TACACTCACTGTTAGTTCTTACCTC (XCR2 MO; 25-mer, underlined sequence is the region in the intron), was designed against the consensus sequence at the first exon-intron boundary derived from the sequence of two distinct XCR2 genomic clones. A standard control morpholino oligo (SC MO) from GeneTools was used as a control.
Cell culture and luciferase assays
Plasmids used for transfection were: pCS2+XNR1
(Lustig et al., 1996);
derrière/CS2+ (Sun et al.,
1999); pCS-mouse nodal and pCS-mouse Cripto-3Flag
(Yeo and Whitman, 2001);
pCS-XCR2-3Flag that was generated by PCR-based subcloning and includes three
repeats of the Flag epitope fused after Val191 of XCR2 in pCS-XCR2;
pCS2-6Myc-mouse FAST1 (FOXH1) (Weisberg et
al., 1998); Mix.2 ARE A3-lux
(Chen et al., 1996); and
pcDNA3.1/V5-His-TOPO/lacZ (Invitrogen). Human embryonic kidney 293T cells were
cultured in 5% CO2 at 37°C in DMEM supplemented with 10% FBS,
100 U/ml of penicillin and 100 µg/ml of streptomycin. Transient
transfection assays were performed by the calcium phosphate method in
triplicate (Sambrook and Russell,
2001). Plasmid mixtures contained 200 ng of each expression
construct, 200 ng of the luciferase reporter plasmid, 80 ng of CMV-ß-gal
plasmid, and various amounts of pCS2+ vector to maintain a constant amount of
total DNA. ß-gal activity was confirmed to vary linearly with the
quantity of plasmid transfected. Luciferase activity was measured 48 hours
post-transfection and was normalized to the activity of co-transfected
ß-galactosidase activity. The fold differences in luciferase activity
were calculated using the luciferase activity of the pCS2-6Myc-mouse FAST1
(FAST1) transfected control as basal level activity. Expression of each
epitope-tagged protein was confirmed by western blotting.
Microinjection, immunohistochemistry and in situ hybridization analyses
Xenopus laevis embryos were obtained by artificial fertilization,
and embryos were staged according to Nieuwkoop and Faber
(Nieuwkoop and Faber, 1956).
Synthetic EGFP mRNA was made using SP6 mMESSAGE mMACHINE (Ambion),
and was co-injected with XCR2 MO and/or various plasmids into the marginal
region of one blastomere of two-cell-stage embryos. Embryos were sorted into
left- or right-injected groups based on EGFP-fluorescence, and then fixed for
in situ hybridization analysis, or scored for heart and gut morphology at
stage 45-46 according to Branford et al.
(Branford et al., 2000).
In situ hybridization analyses were performed according to Sive et al.
(Sive et al., 2000), but with
the addition of 0.3% CHAPS in 2xSCC and 0.2xSSC for probe washing.
In Fig. 6I,K-V,
Fig. 7C,D and
Fig. 8A-K,M-Q, hybridization
and wash steps were performed at 70°C. RNA probes were synthesized from
pCRII-XNR1 and pCRII-XATV. After staining, pigmented embryos were bleached and
cleared. Immunohistochemistry was carried out according to Faure et al.
(Faure et al., 2000) and Lee
et al. (Lee et al., 2001). The
sarcomere myosin-specific antibody MF 20 was provided by the Developmental
Studies Hybridoma Bank at the University of Iowa. The signal was detected with
AlexaFluor594 donkey anti-mouse IgG (H+L) antibody (Molecular Probes), or with
peroxidase-conjugated donkey anti-mouse IgG (H+L) antibody (Jackson
ImmunoResearch), and the DAB Substrate Kit (Vector Laboratories).
|
). Ornithine decarboxylase (ODC)
was used as an internal control. Reverse transcriptase negative (RT-)
reactions were carried against all samples using ODC primers to
confirm the absence of genomic DNA contamination. Primers used for PCR were:
FRL1/XCR1 (up, 5'-CTGGTTTTTGCTAAGGACAC- 3';
down, 5'-TTGCAATGCTTGATAAAATG-3'); for temporal expression
analysis of XCR2 (up, 5'-GCTGCGCATATGGGGTTCTT-3'; down,
5'-CGATAATGCAGCCTTGTTTTCTCT-3'); for RNA splicing analysis of
XCR2 (up, 5'-GCCCTTGGGATCCTTACATT-3'; down,
5'-GAGTCAA- TGTTATAAATATGAAT-3'); XCR3 (up,
5'-GCTGTAATTCGCTTGG-GAAC-3'; down,
5'-TTTTGGACATGCACAGAAGC-3'); VG1 (up,
5'-GACCGCTAACGATGAGTG-3'; down,
5'-AGGAATGTCTTCTGGCTC-3'); XNR1
(Lustig et al., 1996);
ODC
(http://www.xenbase.org/).
PCR products were separated by agarose gel electrophoresiss and visualized by
ethidium bromide staining. PCR products of XCR1 (FRL1),
XCR2, XCR3S and XCR3L were recovered from gels and confirmed
by sequencing.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Tao et al., 2005;
Yabe et al., 2003), we
searched the X. laevis EST database for additional EGF-CFC family
members. Full sequencing of two EST clones of potential novel EGF-CFC family
members identified these as two novel Xenopus EGF-CFC genes,
XCR2 and XCR3 (Fig.
1A). The XCR2 and XCR3 proteins are 34.6% and 30.6% identical to
FRL1, respectively, and 30.6% identical to each other. Examination of the
X. tropicalis EST and genomic databases did reveal the existence of
clear X. tropicalis orthologs for each of the X. laevis
EGF-CFC genes (data not shown). While their overall sequences are quite
divergent from one another, the three X. laevis EGF-CFC proteins
share substantial conservation in the EGF-like and CFC domains, and all
contain a putative O-linked fucosylation site, which has been
identified in mammalian cripto as being crucial for function as a nodal
co-receptor (Schiffer et al.,
2001; Yan et al.,
2002). The three Xenopus EGF-CFC proteins are nearly
identical in size, with the notable exception being that the XCR3
transcript occurs in two alternatively spliced forms; the longer form encodes
72 additional amino acids near the N terminus of the protein
(Fig. 1A). This additional
stretch is cysteine-rich but shows no homology to other known proteins or
domains. The full-length sequences for XCR2, XCR3S (short form) and
XCR3L (long form) have been submitted to GenBank under Accession
Numbers AY796186, AY796188 and AY796189, respectively. XCR2 and
XCR3 have also been identified independently by Dorey and Hill (K.
Dorey and C. Hill, personal communication). To render the nomenclature for
Xenopus EGF-CFC factors consistent we, Dorey and Hill, and the
investigators who originally identified FRL1
(Kinoshita et al., 1995) (M.
Kirschner, personal communication) propose to rename FRL1 as
XCR1.
To begin to characterize the developmental roles of Xenopus
EGF-CFC factors, we examined the temporal expression patterns of XCR1,
XCR2 and XCR3 by RT-PCR (Fig.
1B). Transcripts for all of the XCR genes were detected in
unfertilized eggs, and XCR1 and XCR3 were expressed through
early embryogenesis until the early neurula stage (stage 15). XCR2
was expressed at very low levels maternally, expression increased markedly at
the end of gastrulation (stage 12), and was maintained at high levels through
tadpole stages (stage 45). XCR3 was detected as two bands
corresponding to the two splicing variants, XCR3S and XCR3L,
which showed similar temporal expression patterns. XCR1 and
XCR3 were expressed ubiquitously before gastrulation
(Fig. 2A), whereas
XCR2 expression was too low to compare it in the different regions of
the pre-gastrula embryo (not shown). We next examined the spatial expression
patterns of the three XCR genes by in situ hybridization
(Fig. 2). XCR1
expression was strong in the entire prospective ectoderm at the beginning of
gastrulation, and became restricted to the prospective neural plate by stage
12 (Fig. 2B,C), consistent with
previous reports (Wessely et al.,
2004; Yabe et al.,
2003). XCR3 was strongly expressed in the Organizer and
the prospective endoderm at the onset of gastrulation
(Fig. 2D). This expression
became enriched anteriorly during gastrulation
(Fig. 2E). XCR2 was
expressed at very low levels until stage 12, when its expression was
detectable on the dorsal side (Fig.
2F). This dorsal expression was further restricted to the midline
during neurulation (Fig. 2G),
and in the notochord through neurula stages
(Fig. 2H,I). XCR2 was
also bilaterally expressed in the lateral plate region at stage 20
(Fig. 2H,J). XCR2 was
expressed in the prospective heart region at stage 25, and in the floor plate
and dorsal endoderm in the dorsal midline
(Fig. 2K,M). Dorsal midline
expression decreased by stage 30, whereas the heart region expression was
maintained (Fig. 2L,N). The
markedly different temporal and spatial expression patterns of the XCR genes
suggest that they have distinct developmental roles.
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).
Left-side XCR2 expression is essential for left-right patterning
We next investigated the role of XCR2 in early frog development
using antisense morpholino oligonucleotide (MO)-mediated depletion. Because
preliminary experiments indicated that two antisense MOs targeting
XCR2 translation were ineffective (data not shown), we designed an
antisense MO targeting the first exon-intron junction to block XCR2
pre-mRNA splicing. We cloned genomic DNA containing XCR2 by PCR, and
found the position of the first intron to be conserved with known EGF-CFC
genes (data not shown, GenBank Accession Number AY796187)
(Colas and Schoenwolf, 2000).
A 25 nucleotide antisense MO was designed against the first exon-intron
boundary; three bases targeted the first exon and 22 bases targeted the first
intron (Fig. 4A,B). We first
examined whether the XCR2 MO blocks endogenous RNA splicing by RT-PCR, using
primers that span the first intron. In XCR2 MO-injected embryos, the amount of
mature XCR2 mRNA was decreased and an abnormal splicing variant was
generated from a cryptic splice donor site
(Fig. 4C). Correct splicing of
XCR2 was inhibited through stage 35. Splicing of XCR2 mRNA
was not affected by control MO injection. The abnormal splicing variant
detected in XCR2 MO-injected embryos has an in-frame termination
codon immediately following the end of exon I, resulting in an eight amino
acid long predicted coding sequence (Fig.
4B). These results indicate that this splice-site-targeted
antisense MO is an effective tool for the inhibition of endogenous
XCR2 function.
We next examined the phenotype of XCR2 MO-injected embryos. The XCR2 MO did
not cause any detectable defect in dorsoventral or anteroposterior axis
patterning through stage 45, either in comparison to control MO-injected or
uninjected embryos (Fig. 5A,B). There was also no effect of the XCR2 MO on movement or responsiveness of
swimming tadpoles (data not shown). XCR2 MO-injected embryos were, however,
extensively randomized with respect to left-right axis formation
(Fig. 5C-H). The left-right
patterning defects were scored according to Branford et al.
(Branford et al., 2000); heart
morphology is scored as normal or reversed, and gut morphology is scored for
left or right origin of the gut, and for clockwise or counterclockwise coil
direction. A low dose of XCR2 MO (20 ng) caused 23% reversed and 55% normal
heart formation, 37% reversed and 41% normal gut origin, and 28% reversed and
50% normal gut coil direction (Table
1). A higher dose (50 ng) of XCR2 MO caused complete randomization
of heart asymmetry (37% reversed and 39% normal). By contrast, 50 ng of the
control MO had no significant effect on left-right patterning (90% and 89%
normal heart and gut formation, respectively;
Table 1).
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; Hamada et al.,
2002). The function of EGF-CFC proteins on the right side,
however, has not been specifically addressed. To examine the role of
XCR2 in the left versus the right side of the embryo, embryos were
injected XCR2 MO into one of two blastomeres after the first cleavage, which
divides the embryo along the midline of the future left-right axis. Injection
of 10 ng of XCR2 MO caused complete randomization of heart formation (48%
reversed and 49% normal), and extensive, but incomplete, randomization of gut
formation (23% mirror and 46% normal), when injected on the left side of the
embryo (Table 2). By contrast,
right-side injection did not affect left-right patterning, even at a dose of
20 ng of XCR2 MO (95% and 91% of normal heart and gut formation,
respectively). These results indicate that left-side, but not right-side,
expression of XCR2 is necessary for the correct establishment of
left-right asymmetry. We also examined left-side-specific expression of
XNR1 and XATV in the left LPM to establish whether
XCR2 is required in the left LPM for expression of these genes.
Left-side injection of 10 ng of XCR2 MO inhibited XNR1 expression in
the LPM (84%), whereas right-side injection of XCR2 MO or left-side injection
of control MO had no effect on XNR1 expression
(Fig. 6A-J,
Table 3). Similarly,
XATV expression in the left LPM was inhibited by left-side injection
of 10 ng of XCR2 MO (90%), but the dorsal midline expression of XATV
was not inhibited by XCR2 depletion
(Fig. 6O-R, Table 4).
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;
Lowe et al., 1996). We could
first detect bilateral XNR1 expression at stage 15 in two patches
beside the presumptive notochord region, at the posterior of the archenteron
roof (Fig. 7A-C), and
left-side-specific XNR1 expression began in the LPM at stage 20
(Fig. 7G,H,
Table 8). Bilateral injection
of XCR2 MO did not affect bilateral expression of XNR1 at stage 15,
before left-side-specific XNR1 expression begins
(Fig. 7E-F,
Table 5). At stage 20, the
initiation of expression of XNR1 in the left-side LPM was efficiently
inhibited by XCR2 MO, but the bilateral expression of XNR1 was not
affected (Fig. 7G-J,
Table 5). Left-side-specific
expression of XNR1 was not detectable at stage 18 and 19 (data not
shown). These observations indicate that at post-gastrula stages, bilateral
posterior expression of XNR1 is independent of XCR2, whereas
left-side LPM specific expression is dependent on XCR2 at the earliest stage
at which this expression can be detected.
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). In contrast to injection of wild-type
XCR2 on the right side, which caused ectopic XNR1 expression
in the right LPM and inhibited endogenous XNR1 expression on the left
side in 40% of embryos (Fig.
8B, Table 7),
neither pCS-XCR2 mEGF nor pCS-XCR2 mCFC reversed the asymmetry in
XNR1 expression (Fig.
8A,C,D; Table 7).
These results indicate that the ability of ectopic XCR2 to change the
left-side-specific XNR1 expression depends on the function of XCR2 as
a co-receptor for nodal/VG1 family ligands.
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promoter-driven XNR1 expression plasmid
(pXEX/XNR1) (Sampath et al.,
1997). The heart and gut morphology was inverted by pCS-XCR2
injection into the right side in 57% and 46% of embryos, respectively
(Table 10). Concomitant
injection of 20 pg of pXEX/XNR1 on the left side rescued the
orientation of heart and gut morphology in 79% and 64% of injected embryos,
whereas 20 pg of the pXEX control vector did not. This observation indicates
that ectopic expression of XCR2 on the right is sufficient to initiate
premature, ectopic expression of XNR1 and XATV on the right
side, leading to suppression of the normal expression of these factors on the
left side.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Depletion of XCR2 suppresses XNR1
expression in the left-side LPM, consistent with a role for XCR2 as a
co-factor in XNR1 signaling. The signals that initiate left-side-specific
expression of XNR1 in the frog embryo are not known. One possibility
is that the posterior bilateral expression of XNR1 is functionally
comparable with nodal expression in the peri-nodal region of the
mouse (Brennan et al., 2002;
Saijoh et al., 2003), and that
the XNR1 signal from this posterior region is responsible for the
initiation of XNR1 expression in the left-side LPM through an
XCR2-dependent pathway. A cilia-based mechanism for the left-right asymmetric
distribution of nodal protein has been proposed
(McGrath et al., 2003;
Nonaka et al., 2002;
Nonaka et al., 1998); whether
a comparable mechanism might distribute the posterior-bilateral XNR1
asymmetrically is an interesting possibility, but it requires additional
investigation. Alternatively, an asymmetric distribution of VG1 activity has
been proposed as a mechanism for the initiation of left-right signaling
(Chen et al., 2004;
Hyatt et al., 1996;
Kramer and Yost, 2002). This
signal could also be dependent on XCR2 (alternatively it could depend on XCR1
and XCR3 (K. Dorey and C. Hill, personal communication), and therefore our
data do not distinguish between these possibilities. We found that inhibition
of XCR2 blocks left-side-specific expression of XNR1 as early as this
expression is detectable. Although we cannot rule out the possibility that a
non-XCR2-dependent signal initiates left-side XNR1 expression at a
level not detectable by in situ hybridization, our data are consistent with
the possibility that the initiation of asymmetric XNR1 expression is
XCR2 dependent. Once asymmetric XNR1 expression is initiated,
XCR2 is also likely to be required for the activity of the
FAST1/FOXH1-mediated positive-feedback loop maintaining XNR1
expression in the left LPM.
The bilateral expression of XNR1 in the posterior region of
neurula stage embryos is not affected by depletion of XCR2. While it
is possible that bilateral posterior expression is supported by residual
XCR2, or by the perdurance of XCR1 or XCR3 from
early embryogenesis, it seems likely that this expression is maintained by a
mechanism distinct from the positive-feedback loop of XNR1. The
pattern of XCR2- independent expression of XNR1 is similar
to the expression of mouse Nodal in the peri-nodal region, which is
independent of Cryptic (Gaio et
al., 1999; Yan et al.,
1999), indicating a broad conservation of this posterior pattern
of neurula-stage gene expression of nodal ligands among vertebrates. It is
possible that this posterior bilateral expression of XNR1 is
transferred anteriorly in a left-right asymmetric manner to establish the
asymmetric expression of XNR1 in the LPM
(Wright, 2001), but the basis
for this transition to asymmetry remains unclear.
In contrast to effects on the left side LPM, depletion of XCR2 on
the right side of the embryo does not detectably alter left-right patterning.
Although expression of both nodal family ligands and the nodal antagonists
lefty/antivin is excluded from the right side
(Branford et al., 2000;
Cheng et al., 2000;
Joseph and Melton, 1997;
Lowe et al., 1996;
Tanegashima et al., 2000), the
extent or significance of their potential diffusion across and/or from the
midline is not known. On the one hand, our results indicate that
XCR2-dependent activity of neither the nodal family ligands nor their
antagonists is essential on the right side for correct patterning. On the
other hand, ectopic expression of XCR2 is sufficient to reverse the
left-right polarity, as can ectopic expression of TGFß family ligands or
activated receptors (Chen et al.,
2004; Hanafusa et al.,
2000; Hyatt et al.,
1996; Sampath et al.,
1997; Toyoizumi et al.,
2000). Data from several laboratories, including our own, have
shown that EGF-CFC proteins function as co-factors for nodal/GDF1 signaling,
but are not sufficient to activate SMAD2 signaling in absence of the ligands
(Fig. 3) (Saijo et al., 2000;
Yan et al., 2002;
Yeo and Whitman, 2001;
Reissmann et al., 2001). That
ectopic XCR2 expression on the right side is enough to reverse
left-right polarity indicates, therefore, that there are sufficient levels of
nodal/GDF1 family ligands in the right-side LPM to initiate the left-side
program when an exogenous co-receptor is provided. Ectopic XCR2 is
also sufficient to activate ectopic XNR1 expression in the somites
(Fig. 6), indicating that
ectopic XCR2 can sensitize somites to endogenous nodal-related signals. This
also suggests that the diffusion of XNR1 or related ligands is not restricted
to the LPM at somite stages, although the possibility that nodal ligands are
expressed at undetectable, but functionally significant, levels in the somite
itself cannot be ruled out. Because left-side or right-side specific
injections at the two-cell stage lead to variable expression in midline
structures, our data do not distinguish a function for XCR2 expressed in
midline structures such as the notochord in left-right patterning.
Why ectopic XCR2 expression on the right is sufficient to flip the
left-right orientation of XNR1 expression remains an interesting
theoretical question. This effect is eliminated by point mutations in either
of the two domains required for XCR2 function as a nodal ligand co-receptor,
strongly indicating that it is this function that mediates the observed effect
on axis orientation. That ectopic XCR2 expression is sufficient to
reverse the polarity of XNR1 expression suggests: (1) that XCR2 is
limiting for activity of endogenous nodal/GDF1 family ligands on the right
side of the embryo; (2) that elevated XCR2 enhances the activity of these
ligands more than it enhances the activity of any lefty/antivin antagonists
(Chen and Shen, 2004;
Cheng et al., 2004;
Tanegashima et al., 2004); (3)
that the enhancement of activity of nodal/GDF1 ligands present on the right
side is sufficient to establish a positive-feedback loop for XNR1
expression in the right-side LPM; and (4) that this early XNR1
expression on the right induces XATV, which in turn diffuses to
suppress the normal activation of XNR1 signaling and expression on the left
side.
The establishment of the left-right axis is a fascinating example of how an initial symmetry breaking event establishes a system of feedback loops that maintain a sharply divided asymmetry in the activity of a diffusible signaling molecule, in this case XNR1. Our observations suggest that XCR2 may be a critical limiting component of both the amplitude and the spatial extent of the left-side signal during left-right patterning. Consideration of this role for EGF-CFC factors will therefore be important for theoretical modeling of the dynamics of left-right patterning.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adachi, H., Saijoh, Y., Mochida, K., Ohishi, S., Hashiguchi, H.,
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