1 National Research Institute of Aquaculture, Nansei, Mie 516-0193, Japan
2 Department of Anatomy and Cell Biology, Roy J. and Lucille A. Carver College
of Medicine, University of Iowa, Iowa City, Iowa 52242, USA
3 Laboratory for Vertebrate Axis Formation, Center for Developmental Biology,
RIKEN, Kobe 650-0047, Japan
* Authors for correspondence (e-mail: hibi{at}cdb.riken.jp and suzukitr{at}fra.affrc.go.jp)
Accepted 7 January 2004
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SUMMARY |
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Key words: Left/right asymmetry, Nodal, Cerberus/Dan family, Nodal flow, Zebrafish
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Introduction |
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The L/R-biased signals are thought to be transferred from the node to the
lateral plate mesoderm (LPM), leading to the left side-asymmetric expression
of nodal, lefty and the homeobox gene pitx2 in the LPM.
Nodal is a member of the transforming growth factor-ß (TGF-ß) family
of cytokines and regulates the expression of pitx2 and
lefty, which encodes a feedback regulator for Nodal signaling in the
left LPM (Essner et al., 2000;
Liang et al., 2000
;
Shiratori et al., 2001
;
Yan et al., 1999
;
Yoshioka et al., 1998
). Nodal
also activates nodal expression through an auto-regulation mechanism
that involves the transcription factor FoxHI
(Long et al., 2003
;
Norris et al., 2002
;
Osada et al., 2000
;
Saijoh et al., 2000
). In
zebrafish, Nodal signaling is also involved in the L/R patterning of the
diencephalon (Concha et al.,
2000
; Gamse et al.,
2003
; Liang et al.,
2000
; Long et al.,
2003
). For the initiation of L/R patterning in the mouse, Nodal is
required not only in the LPM, but also near the node
(Brennan et al., 2002
;
Saijoh et al., 2003
).
L/R asymmetry is maintained by midline barriers, which block the transfer
of the left-side determinants. These midline barriers function either within
the organizer itself or within differentiated derivatives of the midline
organizer, such as the notochord and floor plate
(Bisgrove et al., 2000;
Lohr et al., 1997
;
Schlange et al., 2001
). In
zebrafish mutants no tail, floating head and bozozok (also
known as momo) that perturb midline development, there is an increase
in the incidence of the bilateral expression of left side-specific genes in
the LPM (Bisgrove et al., 2000
;
Danos and Yost, 1996
). Loss of
Lefty1, which is expressed in the left floor plate, in mouse is reported to
cause left isomerism (Meno et al.,
1998
).
In zebrafish, there are three known nodal-related genes,
cyclops (cyc), squint (sqt) and
southpaw (spaw). cyc and spaw are
expressed in the left LPM (Long et al.,
2003; Rebagliati et al.,
1998a
; Sampath et al.,
1998
). cyc is also expressed in the left diencephalon, in
a region that corresponds to the prospective parapineal and pineal bodies
(Liang et al., 2000
;
Rebagliati et al., 1998a
;
Sampath et al., 1998
).
spaw is also expressed near Kupffer's vesicle in a similar way to
nodal expression near the node in mouse
(Long et al., 2003
). Mutations
in the cyc gene have a minimal effect on visceral organ asymmetry
(Bisgrove et al., 2000
;
Chen et al., 1997
;
Chin et al., 2000
). However,
several independent lines of evidence implicate Nodal signaling in the
establishment of L/R asymmetry within the zebrafish. Loss of the late zygotic
function of One-eyed pinhead (Oep), an EGF-CFC-family protein required for
Nodal signaling, leads to defects in left-side gene expression and in visceral
organ and diencephalic laterality (Gamse
et al., 2003
; Liang et al.,
2000
; Yan et al.,
1999
). Mutations in schmalspur (sur), which
encodes FoxH1 and mediates Nodal signaling, also lead to defects in L/R
patterning (Bisgrove et al.,
2000
; Chen et al.,
1997
; Pogoda et al.,
2000
; Sirotkin et al.,
2000
). Finally, antisense morpholino (MO)-mediated inhibition of
Spaw disrupts the left-side expression of cyc, pitx2, lefty1 and
lefty2 and leads to defects in L/R patterning in visceral organs and
in the diencephalon (Long et al.,
2003
). sqt is not expressed asymmetrically, but in the
absence of sqt, asymmetric expression of spaw is disrupted
(Long et al., 2003
). In sum,
all of these data strongly implicate Nodal signaling in L/R patterning in the
zebrafish, with spaw having a major role within the LPM and in at
least the initial steps of diencephalic asymmetry.
Members of the Cerberus/Dan family have been implicated in L/R patterning
by virtue of the asymmetric expression patterns of some of these proteins. In
chick, for instance, caronte exhibits left-side expression within the
paraxial mesoderm and LPM (Rodriguez
Esteban et al., 1999; Yokouchi
et al., 1999
). Thus, Caronte has been postulated to transmit a
signal from the node to the left LPM. Caronte can function as an inhibitor of
BMP signaling (Rodriguez Esteban et al.,
1999
; Yokouchi et al.,
1999
). However, it was reported that BMP signaling positively
regulates nodal expression in the left LPM by inducing an EGF-CFC
protein that is required for the LPM's competence to respond to Nodal ligands
(Fujiwara et al., 2002
;
Piedra and Ros, 2002
;
Schlange et al., 2002
;
Schlange et al., 2001
),
raising the possibility that Caronte has functions other than inhibiting BMP
signaling. Another member of the Cerberus/Dan family, Dante, is expressed
around the node in mouse (Pearce et al.,
1999
). A role of Dante in L/R patterning has not yet been
established.
Here we report the isolation of a novel gene, named charon, that encodes a Cerberus/Dan family secreted protein. charon is expressed from the early segmentation stages in the region embracing Kupffer's vesicle, adjacent and medial to the bilateral (`perinodal') spaw-expression domains. We found that Charon inhibits the activities of the Nodal-related proteins and identified Southpaw as a physiological target. Specifically, our data indicate that the antagonistic interaction between Charon and Nodal (Southpaw) plays an important role in L/R patterning in zebrafish.
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Materials and methods |
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Database searching and molecular cloning
Four distinct Fugu and zebrafish genes homologous to chick
caronte were found in the NCBI database and in the Fugu
(Fugu rubripes) genome database of the Doe Joint Genome Institute
(http://fugu.jgi-psf.org)
by a BLAST search. Three of these genes encoded proteins with strong homology
to Cerberus/Caronte, PRDC and Gremlin. The other was distantly related to any
of the known Cerberus/Dan family proteins. Here, we describe the isolation of
Fugu, zebrafish and flounder cDNAs of the gene charon, whose
protein displayed the strongest homology to Cerberus/Caronte. The Fugu
charon cDNA containing the whole open reading frame (ORF) was amplified
by PCR with the following primer set: sense,
5'-CGGGATCCCAGACGACAATTTTCCTGTTG-3', and antisense,
5'-CCATCGATGCAGGCGTCCCGAAGCTGCGT-3'. The resulting fragment was
cloned into pBluescript II (pBS-fugu-charon). The cDNA fragment of zebrafish
charon was isolated from a segmentation stage (15 hours
post-fertilization, 15 hpf) cDNA library, which was constructed using a
Marathon cDNA library synthesis kit (Clontech), with 5' and 3'
RACE using the primers 5'-GGTTTCACACTTGCACTCCTCAACG-3' and
5'-GCACTCCTCAACGATCAGTACGCACC-3' for 5' RACE, and
5'-CAGCGCATAACGGAGGAGGGCTGTG-3' and
5'-GGAGGGCTGTGAGACGGTGACCGTT-3' for 3' RACE. The resulting
fragment was subcloned into pDrive (Qiagen) (pDrive-zcharon for the 5'
RACE clone). After determining the 5'- and 3' ends of the
full-length charon cDNA, the zebrafish charon ORF was amplified by
PCR with the following primers: sense,
5'-CGGGATCCCGAAACCTTGAACCGCAAGATT-3', and antisense,
5'-CCATCGATGTAAATTAAACATATCTGTGTT-3'. The resulting fragment was
cloned into pCS2MT or pCS2 (pCS2MT-zcharon or pCS2-zcharon). A part of the
flounder charon cDNA was obtained by PCR with primers that
corresponded to the conserved amino acids RVTAAGC and ETGREEK: sense,
5'-AGCGTGTGACGGCGGCGGGATG-3', and antisense,
5'-CCTTTTCCTCGCGGCCTGTTTC-3'. The obtained fragment was verified
by sequencing. Using this fragment as a probe, a putative full-length clone of
flounder charon was isolated from a lambda ZipLox cDNA library of
20-somite flounder embryos, and the lambda phage clone was converted to the
plasmid (pZL-fl-charon). The nucleotide sequences of zebrafish charon,
Fugu charon and flounder charon were deposited in the DDBJ
databank under accession numbers AB110416, AB110417 and AB1100418,
respectively. Zebrafish PRDC and gremlin will be published elsewhere.
Constructs, RNA synthesis and transcript detection
pcDNA3.1-Charon-Myc was constructed by insertion of the Charon-Myc fragment
(a Myc tag in the carboxy terminal) from pCS2MT-zcharon into the
BamHI and EcoRI sites of pcDNA3.1. pcDNA3.1-PRDC-Myc was
constructed in a similar manner to that for pcDNA3.1-Charon-Myc.
pcDNA3.1-HA-Spaw was constructed by insertion of the
EcoRI-NotI fragment from pCS2+ActHASpaw
(Long et al., 2003) into
pcDNA3.1. pBS-fugu-charon was used for in situ hybridization of the
Fugu embryo. A DIG-labeled riboprobe was made with T3 RNA polymerase
(Promega) after NotI digestion. pZL-fl-charon was used for the
flounder embryo. A DIG-labeled riboprobe was made with SP6 RNA polymerase
(Promega) after SalI digestion. The zebrafish riboprobe was made with
BamHI-digested pDrive-zcharon using SP6 RNA polymerase (Promega).
Synthetic zebrafish charon RNA was produced with pCS2MT-zcharon or
pCS2-zcharon. After NotI digestion, the RNA for Myc-tagged Charon or
untagged Charon was transcribed in a solution containing an RNA cap structure
analog (New England BioLabs) and SP6 RNA polymerase (Promega). Synthesis of
RNAs for cyc, sqt and spaw was performed as described
previously (Long et al., 2003
;
Rebagliati et al., 1998a
;
Rebagliati et al., 1998b
). The
method for detecting spaw, cyc, ntl, goosecoid, six3.2, pax2.1,
nkx2.5 and cardiac myosin light chain (cmlc2)
expression was also previously published
(Chen and Fishman, 1996
;
Hashimoto et al., 2000
;
Long et al., 2003
;
Yelon et al., 1999
).
Whole-mount in situ hybridization and two-color staining were performed as
described (Hashimoto et al.,
2000
; Long et al.,
2003
; Long and Rebagliati,
2002
).
Interaction assay
COS7 cells in a 10 cm-diameter dish (approximately 106 cells)
were transfected with 10 µg of pcDNA3.1-Charon-Myc, pcDNA3.1-PRDC-Myc or
pcDNA3.1-HA-Spaw by a standard calcium phosphate precipitation method. After
20 hours, the medium was changed from 10% fetal calf serum-contained DMEM to
serum-free Opti-MEM1 (Invitrogen). After 48 hours, the supernatants were
harvested. The supernatants containing Charon-Myc (25 µl), PRDC-Myc (25
µl) or HA-Spaw (250 µl) were mixed in a combination described in
Fig. 5 and incubated with
anti-Myc (9E10, Invitrogen) or anti-HA (3F10, Roche) antibodies, and protein G
sepharose, at 4°C for 15 hours. The precipitates were washed five times
with washing buffer: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100,
proteinase inhibitor Complete Mini EDTA-free (Roche), eluted with Laemmli's
sodium dodecyl sulfate (SDS) loading buffer, and separated on an SDS-4 to 20%
gradient polyacrylamide gel. The immune complexes were visualized by a
chemiluminescence system (Western Lightning; PerkinElmer Life Sciences).
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Results |
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charon expression
charon transcripts were first detected at the beginning of
somitogenesis (2-3 somite stages) in the hypoblast of the tailbud region in
zebrafish (Fig. 2A,B). At the
10-somite stage (14 hpf), the expression domain was observed as a
horseshoe-shaped zone (with the anterior side open) in the tail region
(Fig. 2C,D). Sagittal
sectioning revealed that the charon-expressing cells were either
within the epithelial lining of Kupffer's vesicle or very closely apposed to
Kupffer's vesicle (Fig. 5M).
The expression was strongest at the 10-somite through to the 14-somite stage
(16 hpf) (Fig. 2G,H), then
gradually disappeared, and it was not detected at 24 hpf. charon was
not detected anywhere besides the region adjacent to Kupffer's vesicle at any
developmental stage. charon was expressed in the region adjacent to
Kupffer's vesicle in Fugu and flounder embryos, as in zebrafish
(Fig. 2I-L).
We next examined the regulation of charon expression using the
zebrafish mutants boz, ntl, cyc, sqt and oep. boz mutant
embryos display variable defects in dorso-axial structures including the
dorsal forerunner cells, which give rise to Kupffer's vesicle
(Fekany et al., 1999). In
boz mutant embryos, charon expression was reduced or not
detectable, with a variable penetrance
(Fig. 3B,C). The
charon expression was not detected in the ntl mutant embryos
(Fig. 3D), which display
defective development of the dorsal forerunner cells, notochord and tail
(Melby et al., 1996
). The
charon expression was not affected in the cyc mutant embryos
(Fig. 3G), but it was reduced
or absent in the sqt mutant embryos, which exhibit defective
development of Kupffer's vesicle (Dougan
et al., 2003
) (Fig.
3E,F). In the oep mutant embryos, the charon
expression was comparable to that in the wild-type embryos, but the expression
domain was smaller than that in the wild-type embryos, in proportion to the
size of Kupffer's vesicle (Fig.
3H). The charon expression was not detected in embryos
injected with a large amount of RNA for the Nodal/Activin inhibitor Atv/Lefty1
(Thisse and Thisse, 1999
)
(Fig. 3I). The
atv/lefty1 RNA-injected embryos did not have Kupffer's vesicle (data
not shown). The data suggest that charon expression depends on the
formation of the dorsal forerunner cells/Kupffer's vesicle, which is dependent
on the Nodal signaling.
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charon and spaw are expressed in an adjacent region near Kupffer's vesicle
Charon inhibited all the Nodal-related proteins in zebrafish in the
misexpression studies. To address which Nodal-related ligand(s) is the
physiological target for Charon, we first compared the expression profiles of
charon and the nodal-related genes. sqt expression
did not overlap with charon expression at any developmental stage
(data not shown). cyc is expressed in the tailbud region at the bud
stage, but its expression disappears by the 2-3 somite stage
(Rebagliati et al., 1998a;
Sampath et al., 1998
),
indicating that the expression of cyc coincides with that of
charon spatially but not temporally. spaw displays a similar
expression to charon in the tailbud region
(Long et al., 2003
);
spaw expression is first detectable at the 4-6-somite stage (14 hpf),
slightly later than the initial charon expression. The
spaw-expressing cells were located bilaterally in two domains
flanking (or possibly in cells lining) Kupffer's vesicle
(Long et al., 2003
)
(Fig. 5H-J). During
somitogenesis, spaw and charon continued to be expressed in
adjacent regions close to Kupffer's vesicle
(Fig. 5K-M). Two-color staining
and examinations of cross-sectioned embryos revealed that the
spaw-expressing cells were located dorsal and lateral to the
charon-expressing cells (Fig.
5N-Q). The expression domains of spaw and charon
did not overlap. However, both genes encoded secreted proteins, so their
domains could overlap at the level of protein. The expression of both genes
was maintained until the end of somitogenesis (data not shown). These data
indicate that Spaw is the best candidate for a functional target for
Charon.
Knockdown of Charon leads to a defect in heart positioning
We performed loss-of-function experiments by injecting antisense MOs
against the translational initiation site of charon
(charon-MO, Fig. 6).
To examine the specificity of charon-MO, we co-injected
charon-MO with charon RNA into embryos. The phenotypes
caused by the misexpression of charon were suppressed by the
co-injection of charon-MO (Fig.
6A,B, Table 1).
Embryos injected with charon-MO alone (charon morphant
embryos) did not show any gross morphological abnormalities during early
development and survived for at least 1 week after hatching
(Fig. 6L). However, we found
that the charon morphant embryos displayed abnormal positioning of
the heart (Fig. 6C-K,
Table 2). The process of heart
positioning can be divided into two steps, jogging and looping
(Chen et al., 1997); jogging
and looping are two aspects of heart L/R asymmetry. Wild-type embryos display
a leftward shift (jog) of the heart tube from approximately 26-30 hpf and
rightward (D-) looping from 30-60 hpf (Chen
et al., 1997
) (Table
2). The expression of the homeobox gene nkx2.5 and a
cardiac-specific myosin light chain (cmlc2) gene can be used
to visualize the jogging and looping of the heart
(Chen and Fishman, 1996
;
Yelon et al., 1999
). Using
these markers, we found that the laterality of the heart jogging was severely
perturbed in the charon morphant embryos, but not in the control
morphant embryos (Fig. 6C-H,
Table 2). Likewise, heart
looping was disrupted in charon morphant embryos, as shown by
significant increases in the fraction of embryos with reversed heart loops
(L-loops) or unlooped hearts (Fig.
6I-K, Table 2).
These data indicate that Charon is involved in L/R-biased heart positioning
during heart formation.
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Inhibition of Southpaw by Charon is required for the L/R patterning
Left-side expression of spaw, pitx2, atv/lefty1 and
lefty2 is lost in embryos with reduced Spaw activity
(Long et al., 2003). The same
effects are seen in mouse mutant embryos that lack nodal expression
in the node (Brennan et al.,
2002
; Saijoh et al.,
2003
), suggesting a role for Spaw and Nodal in the initial
left-side determination. Because charon was co-expressed with
spaw in the tail region and Charon inhibited Spaw's function in the
misexpression study, we thought it probable that the antagonistic interaction
between Charon and Spaw in the tail region plays a role in L/R patterning. To
address this, we conducted an epistatic analysis using the MOs for
spaw and charon. We examined the expression of pitx2,
atv/lefty1, lefty2 and cyc in embryos injected with both
spaw-MO and charon-MO, or charon-MO alone.
Injection of 0.8 ng of charon-MO occasionally increased the frequency
of embryos expressing pitx2, cyc, atv/lefty1 or lefty2 on
the right side, but most often led to bilateral expression of these genes in
the LPM and diencephalon (for pitx2, cyc and atv/lefty1)
(Fig. 8B,E,H,
Table 3). Coinjection of 8 ng
of spaw-MO with the charon-MO reduced or abolished the
expression of these genes on both the left and right sides
(Fig. 8C,F,I,
Table 3). Because the same
effect is observed in the spaw morphant embryos
(Long et al., 2003
), we
conclude that spaw is epistatic to charon in the expression
of the left-specific genes. These data provide additional evidence that Spaw
functions as a left-side determinant and, more importantly, indicate a
functional interaction between Charon and Spaw in the L/R patterning.
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Discussion |
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One of the Cerberus/Dan family proteins, mouse dante, is expressed
in the definitive node from the bud stage
(Pearce et al., 1999).
dante expression in the mouse node is very similar to charon
expression in the Kupffer's vesicle region of the zebrafish, suggesting that
Dante is an orthologue of Charon. However, only a partial sequence of
dante has been reported and its biochemical function is not yet
known, although it has been suggested that Dante functions as a Nodal
inhibitor (Brennan et al.,
2002
). Thus, the relationship between Charon and Dante remains to
be elucidated. Future comparison between the zebrafish charon
morphant embryos and dante-deficient mouse embryos, and biochemical
analysis of Dante activity against Nodal will clarify this point.
In the mouse, dante and nodal are initially expressed
symmetrically in the node region, and the dante expression becomes
stronger on the right side of the node by early somitogenesis, in contrast to
the stronger expression of nodal on the left side
(Collignon et al., 1996;
Pearce et al., 1999
). We
observed that the majority of embryos showed symmetric expression of
charon (Fig. 2),
implicating that the regulation of charon is different from that of
dante in mouse. However, it could not be excluded that
charon shows right-biased expression transiently.
Atv/Lefty1 and Lefty2, members of TGF-ß family, function as feedback
inhibitors for Nodal signaling (Cheng et
al., 2000; Meno et al.,
1999
; Sakuma et al.,
2002
). atv/lefty1 is expressed in the Kupffer's vesicle
region during early somitogenesis, and its expression is abrogated in
oep mutant embryos (Bisgrove et
al., 1999
), suggesting that the atv/lefty1 expression in
the Kupffer's vesicle region depends on Nodal signaling. The charon
expression in the Kupffer's vesicle region was dependent on the Nodal
signaling. The expression of charon was abolished or strongly reduced
in the sqt and atv/lefty1-injected embryos
(Fig. 3), which display
defective development of Kupffer's vesicle
(Dougan et al., 2003
). The
charon expression was not affected in the cyc embryos, and
was reduced in the oep embryos, with the severity of the decrease
correlating with the reduction in the size of Kupffer's vesicle. These data
suggest that the charon expression depends on the formation of
Kupffer's vesicle and that Nodal signaling indirectly regulates the
charon expression. Consistent with this, the charon
expression was abrogated or strongly reduced in the boz and
ntl mutant embryos (Fig.
3), which have defects in the formation of Kupffer's vesicle
(Fekany et al., 1999
;
Melby et al., 1996
). However,
because the aforementioned mutants also affect other aspects of mesendoderm
development, we cannot rule out the possibility that the charon
expression is regulated by a signal distinct from those that induce
morphological development of Kupffer's vesicle. Our data indicate that Charon
functions to restrict laterality near Kupffer's vesicle, probably in
cooperation with Atv/Lefty1.
Antagonistic interactions between Charon and Southpaw
Three lines of evidence argue that Southpaw is a physiological target for
Charon. First, Charon interacted with Spaw biochemically and the dorsalizing
activity of Spaw was inhibited by the overexpression of Charon
(Fig. 5). Second, the
expression domains of charon and spaw were in close
proximity to each other in the tail region
(Fig. 5), and both genes encode
secreted proteins that will probably be secreted into overlapping regions.
Third, the loss-of-function experiments showed that Spaw is epistatic to
Charon in the expression of the left side-specific genes
(Fig. 8,
Table 3) in both the LPM and
diencephalon. All of these data suggest that the inhibition of Spaw by Charon
is involved in the generation of embryonic L/R asymmetry. The inhibition of
Spaw's function leads to a reduction or loss of left side-specific gene
expression (Long et al.,
2003), suggesting that Spaw functions as a left-side determinant,
as proposed for Nodal in mouse (Hamada et
al., 2002
). However, spaw is expressed not only in the
Kupffer's vesicle region but also in the left LPM. The functional relevance of
Spaw in the Kupffer's vesicle region has not been tested yet, as the
spaw-MOs would block Spaw activity in all the spaw
expression domains. In this study, Charon, which is expressed in Kupffer's
vesicle, interacted functionally with Spaw, suggesting that Spaw may function
in the Kupffer's vesicle region to initiate left-side determination. However,
there is one significant argument against this idea: in ntl mutants,
spaw expression domains in Kupffer's vesicle are lost, but
spaw is expressed bilaterally in the LPM
(Long et al., 2003
),
suggesting that Spaw in the Kupffer's vesicle region is not required for the
expression of Spaw in the LPM. The situation is different from nodal
in the mouse; elimination of nodal in the node region disrupts
nodal expression in the left LPM in the mouse
(Brennan et al., 2002
;
Saijoh et al., 2003
).
There are several possible explanations for this discrepancy. First, a low
level of Spaw could be transiently expressed in the Kupffer's vesicle region
in the ntl mutant embryos and induce spaw expression in the
LPM in the absence of the midline barriers and the Nodal inhibitor Charon.
Second, if Nodal-class proteins turn over slowly, then pre-existing Nodal
proteins, such as Cyc and Sqt, might compensate for the loss of Spaw in the
Kupffer's vesicle region in the ntl mutant embryos. Consistent with
this possibility, in ntl mutants, cyc expression is lost in
the tailbud but is concomitantly upregulated or shifted into posterior-lateral
territories at the bud stage (Rebagliati
et al., 1998a). Charon could inhibit the dorsalizing activity of
Cyc (Fig. 5). We could not
exclude the possibility that there is a non-Nodal factor, possibly another
TGF-ß, which is expressed in the Kupffer's vesicle region and activates
Nodal signaling in the LPM in the ntl mutant. In mouse, GDF1
expression in the node is required for the expression of nodal,
lefty2 and pitx2 in the left LPM
(Rankin et al., 2000
). It is
also possible that, in the absence of a midline barrier and Charon,
spaw could be expressed in the LPM by a constitutive or default
mechanism.
Role for Charon in L/R asymmetry
Loss of the Charon function by the morpholino-mediated inhibition led to
bilateral expression of the left side-specific genes and randomization of
heart jogging and looping (Figs
6,
7,
Table 2). In this sense, the
phenotypes of the charon morphant embryos are similar to those
observed in zebrafish mutants boz (momo), ntl and
flh, which have defects in the formation of midline structures
(Bisgrove et al., 2000;
Long et al., 2003
), and in
mutant mice deficient in lefty1, which is expressed in the left floor
plate (Meno et al., 1998
),
raising the question as to whether Charon is involved in the formation of `the
midline barrier'. However, the development of midline tissues, such as
notochord and floor plate, and the expression of ntl, shh and
atv/lefty1 were not affected in the charon morphant embryos
(Fig. 7), suggesting that
Charon is dispensable for the formation of the midline barrier. We cannot
completely rule out the possibility that Charon is involved in expression or
function of an unidentified component(s) of the midline barrier. Because
charon is expressed only in the Kupffer's vesicle region, we favor
the hypothesis that Charon functions near Kupffer's vesicle rather than
regulating midline barrier functions of the notochord or floor plate.
lr dynein-related is expressed in the dorsal forerunner cells, and
cells within Kupffer's vesicle have a cilium, in zebrafish
(Essner et al., 2002),
suggesting that Kupffer's vesicle is equivalent to the node cavity in mouse.
Likewise, the fact that asymmetric expression of the nodal-related
gene spaw begins near Kupffer's vesicle, and that this pattern is
disrupted by mutations that affect forerunner cells, also supports this
equivalence (Long et al.,
2003
). It has not yet been reported whether the cilia in the
Kupffer's vesicle rotate and generate the leftward flow (nodal flow) in
zebrafish. It has also not yet been shown whether the L/R signal is initiated
in Kupffer's vesicle in zebrafish. Here, we have demonstrated that
charon is expressed in or next to Kupffer's vesicle and that Charon
functions in the L/R patterning without affecting the development of the
midline tissue and atv/lefty1-dependent midline barrier, suggesting
that L/R patterning is initiated in the Kupffer's vesicle region. Given that
loss of molecular components of the mouse node and zebrafish Kupffer's vesicle
can result in similar L/R defects, our data provide support for the proposal
that these structures are functionally equivalent and that they have an
evolutionarily conserved role in L/R patterning
(Essner et al., 2002
). It is
tempting to speculate that Nodal-related molecule(s) such as Spaw (or possibly
Cyc) function in the Kupffer's vesicle region in zebrafish in the same way as
had been proposed for Nodal protein in the perinodal region in mice. In this
scenario, the Nodal signaling is biased to the left side of the Kupffer's
vesicle region in zebrafish. Charon might be involved in creating an `all or
nothing' condition of Nodal signaling on each side of the Kupffer's vesicle
region by reducing the Nodal signaling baseline.
In this study, we isolated Charon, a novel player in L/R patterning, and found antagonistic interactions between Charon and Nodal (Southpaw) to be involved in L/R patterning. This finding sheds new light on the role of Cerberus/Dan-related proteins in the process by which the L/R-biased signal is created during early vertebrate development.
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ACKNOWLEDGMENTS |
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Footnotes |
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