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First published online 3 October 2007
doi: 10.1242/dev.009464
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1 Developmental Genetics Group, Graduate School of Frontier Biosciences, Osaka
University, and CREST, Japan Science and Technology Corporation (JST), 1-3
Yamada-oka, Suita, Osaka 565-0871, Japan.
2 Department of Developmental Biology, Center for Integrated Research in
Science, Faculty of Medicine, Shimane University, 89-1 Enyacho, Izumo
693-8501, Japan.
3 Department of Biosignaling and Radioisotope Experiments, Center for Integrated
Research in Science, Faculty of Medicine, Shimane University, 89-1 Enyacho,
Izumo 693-8501, Japan.
4 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.
5 Department of Cell Biology, Research Institute for Microbial Diseases, Osaka
University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
6 Department of Neurobiology and Anatomy, Program in Human Molecular Biology and
Genetics, and The Eccles Program in Human Molecular Biology and Genetics,
University of Utah, Salt Lake City, UT 84112-5330, USA.
Author for correspondence (e-mail:
hamada{at}fbs.osaka-u.ac.jp)
Accepted 20 August 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Left-right asymmetry, Node, Nodal, Extracellular matrix, Glycosaminoglycan, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Shiratori and
Hamada, 2006). We (Saijoh et
al., 2003) and others (Brennan
et al., 2002) have shown that mice deficient in Nodal
expression at the node fail to express Nodal in the LPM. However, it
has been unclear how Nodal produced at the node directs Nodal
expression in the LPM. Previous observations that Nodal is able to act over a
long distance (Chen and Schier,
2001; Yamamoto et al.,
2003) and that Nodal expression is regulated by
Nodal-responsive enhancers (Norris et al.,
2002; Saijoh et al.,
2000) suggest that Nodal produced at the node may travel from the
node to the LPM. However, direct evidence for such transport of Nodal has
proved elusive.
Sulfated glycosaminoglycans (GAGs) have been shown to contribute to
morphogen gradient formation (Hacker et
al., 2005; Lin,
2004). Sulfated GAGs comprise repeating sulfated disaccharides
including N-acetylglucosamine and glucuronic or iduronic acid for
heparin and heparan sulfate (HS), or N-acetylgalactosamine and
glucuronic acid for chondroitin sulfate (CS). HS and CS are covalently linked
to serine residues of membrane-tethered core proteins, giving rise to heparan
sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans,
respectively, that are localized to the cell surface or extracellular matrix
(ECM). The TGF-ß homolog Decapentaplegic (Dpp) acts as an HS-dependent
morphogen in anteroposterior patterning of the Drosophila wing
(Belenkaya et al., 2004); Dpp
is thus not able to traverse mutant cells that lack either an HS-polymerizing
enzyme (sulfateless) or a glypican member of the HSPGs
(dally). Given that Dpp possesses an affinity for heparin
(Groppe et al., 1998), Dpp
molecules secreted into the ECM may be immediately captured by sulfated GAGs
and transported from one GAG chain to another toward more distally located
target cells. Although Nodal is able to exert its action over a long range,
the determinants of this signaling range are unknown.
We have now investigated the mechanism by which the Nodal signal is transferred from the node to the LPM in the mouse embryo. Our results support a model in which Nodal produced by the perinodal cells is directly transported to the LPM via sulfated GAGs that are specifically localized to the basement membrane-like structure between the node and the LPM.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Yan et al., 1999). Enhancer
analysis with lacZ reporter transgenes
(Saijoh et al., 2005) revealed
that an 11 kb genomic fragment of Cryptic (from -10 to +1 kb relative
to the translational initiation site) conferred LPM-specific
ß-galactosidase activity and was thus designated an LPM-specific enhancer
(LPE). The LPE and hsp68 promoter were linked to Cryptic
cDNA, an internal ribosomal entry site (IRES) and lacZ. Two
transgenic lines (#15, #22) that showed LPM-specific expression of this
transgene were established in C57BL/6xC3H F1 mice.
|
); the
hsp68 promoter; Nodal cDNA containing the nucleotide
sequence for the 3xMyc epitope tag (Sigma) positioned four amino acids
downstream from the cleavage site; an IRES; and lacZ. The 40 amino
acid sequence at the NH2-terminal end of the mature
3xMyc-Nodal protein is thus
HHLPEQKLISEEDLNEQKLISEEDLNEQKLISEEDLGDRS, with italics indicating the
3xMyc epitope tag. Two transgenic lines (#35, #133) that showed
node-specific expression were used in this report.
Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed according to standard
procedures with digoxigenin-labeled riboprobes specific for Nodal
(Lowe et al., 1996),
Lefty1 and Lefty2 (Meno
et al., 1997; Meno et al.,
1996), Pitx2 (Meno et
al., 1998), Gdf1
(Rankin et al., 2000) and
Cryptic (Shen et al.,
1997).
Whole-embryo culture
Injection of expression vectors for Nodal or green fluorescent protein
(GFP) and detection of fluorescence were preformed as described previously
(Nakamura et al., 2006). In
some experiments, embryos were exposed to 15 mM sodium chlorate (Kishida
Chemicals), 1 mM p-nitrophenyl ß-D-xylopyranoside
(Sigma) with 0.1% dimethyl sulfoxide as a solubilization carrier, recombinant
mouse Nodal (50 µg/ml, R&D Systems) or recombinant human Activin (400
ng/ml, R&D Systems). For protein injection experiments, approximately 0.01
µl culture medium containing 50 µg/ml bovine serum albumin, 50 µg/ml
recombinant Nodal or 400 ng/ml Activin was injected with a glass needle
(Dramond) and injector (Narishige) into LPM immediately below the endoderm
layer. It should be noted that the same concentration was used to bathe the
embryos as was used in the injection needle.
Immunofluorescence analysis and transmission electron microscopy
Embryonic day 8.0 ICR or transgenic embryos (with or without subsequent
culture) were fixed overnight at 4°C with 4% paraformaldehyde and
cryosectioned at a thickness of 6 µm. Immunofluorescence analysis for GAGs
and Alcian Blue staining were performed as described previously
(Garcia-Garcia and Anderson,
2003; Morriss-Kay and Crutch,
1982), after incubation with heparitinase III (50 mU/ml) or
chondroitinase ABC (5 U/ml, both from Seikagaku) at 37°C for overnight
under commercially recommended conditions. For immunofluorescence analysis of
3xMyc-tagged Nodal, frozen sections were autoclaved at 121°C for 5
minutes in 10 mM sodium citrate buffer (pH 6.0) before exposure to mouse
monoclonal antibodies to Myc (9E10, Santa Cruz Biotechnology) and rabbit
polyclonal antibodies either to laminin (Sigma), ZO-1 (Zymed) or
ß-galactosidase (Zymed); immune complexes were detected with
AlexaFluor-conjugated secondary antibodies (Molecular Probes) and nucleus was
stained with 4',6-diamidino-2-phenylindole (DAPI). For immunoenzymatic
detection of 3xMyc-Nodal by transmission electron microscopy (TEM),
immune complexes were stained with biotinylated antibodies to mouse
immunoglobulin G (Jackson Immunolaboratories), horseradish
peroxidase-conjugated streptavidin (Vector Laboratories), and
p-dimethylaminoazobenzene solution containing 60 mM nickel chloride.
Sections were first examined with a light microscope and subsequently
ultrathin sections were observed with a transmission electron microscope
(EM-002B, Topcon) at a magnification of x1000, x4000 or
x6000. The details of this procedure were described previously
(Hirose et al., 1990).
Interaction between Nodal and sulfated GAGs
Recombinant mouse Nodal (1 µg, R&D Systems) was dissolved in 1 ml of
20 mM HEPES-NaOH (pH 7.2) and incubated overnight at 4°C with 10 µl
heparin-sepharose CL-6B (Amersham). The resin was washed three times with the
HEPES buffer and then suspended sequentially in 100 µl of elution solution
containing various concentrations of NaCl, heparin (porcine intestinal mucosa,
Sigma), CS (bovine trachea, Sigma) or HS (porcine intestinal mucosa, Sigma)
dissolved in the HEPES buffer. The eluate fractions were boiled in SDS sample
buffer containing 0.3 M dithiothreitol and subject to immunoblot analysis with
rabbit antiserum specific for the mature domain of mouse Nodal. The amount of
eluted Nodal was quantified by comparison with recombinant mouse Nodal as a
standard and was normalized relative to the maximal value.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
suggests that excessive Nodal signal on the both sides of the LPM would result
in bilateral Nodal expression in the LPM. We recovered mouse embryos
at the one-somite stage and cultured them in medium with or without an excess
of recombinant Nodal or Activin protein (which would be way above the
concentration of Nodal in the node in vivo) for 7 to 8 hours, until they had
developed to the six-somite stage. Under normal culture conditions,
Nodal expression in the LPM begins at the three-somite stage and has
expanded fully by the six-somite stage. However, exogenous Nodal or Activin
failed to induce Nodal expression in the right LPM (0/10 or 0/9
embryos, respectively; Fig.
2B-D). By contrast, to experimentally generate a situation in
which Nodal can signal through the endoderm, we injected them (at the same
concentrations as those used for bath application) into the right LPM
immediately below the endoderm layer at the one-somite stage
(Fig. 2E). The injected embryos
were cultured without Nodal or Activin and then examined for Nodal
expression in the LPM. Local injection of Nodal or Activin induced ectopic
Nodal expression in the right LPM (11/11 or 2/3 embryos,
respectively; Fig. 2G,H),
whereas injection of bovine serum albumin failed to do so (0/5 embryos;
Fig. 2F). These results thus
indicated that the Nodal signal is not transmitted through the endoderm to the
LPM, although they do not prove that Nodal can travel through the mesoderm
layer. Consistent with the notion, while the endoderm expresses ALK4
and ActRIIB, it fails to express FoxH1 and Cryptic
(also known as Cfc1), an essential component of Nodal signaling (see
Fig. S1 in the supplementary material; data not shown), supporting that the
endoderm is not competent to transmit a Nodal signal.
The Nodal signal travels directly from the node to the LPM
Although our results are consistent with the idea that the Nodal signal is
transferred from the node to the LPM via the internal route, it remained
unclear whether the signal transfer is direct or indirect. Nodal produced at
the node may be directly transported to the LPM
(Fig. 1B), where it activates
Nodal expression through the Nodal-responsive enhancers ASE and LSE
(Saijoh et al., 2003;
Saijoh et al., 2005).
Alternatively, Nodal produced at the node may bind to its receptor and
co-receptor in the node or paraxial mesoderm and thereby generate a secondary
signal that subsequently travels to the LPM and induces Nodal
expression (Fig. 1C). To
distinguish between these possibilities, we focused on Cryptic, a member of
the EGF-CFC family of proteins that serves as a co-receptor for Nodal
(Shen et al., 1997).
Cryptic is expressed specifically in the node, LPM and floor plate
but not in the paraxial mesoderm (Fig.
3D), and it is the only EGF-CFC family gene that is expressed in
those regions of E8.0 embryo; Cripto is not expressed at this stage
except for in the heart tube (see Fig. S2 in the supplementary material).
Furthermore, phosphorylated Smad2/3 is detected in the perinodal cells of the
wild-type embryo, but is absent in the Cryptic-/- embryo,
suggesting that Nodal signal in the node strictly depends on Cryptic (A.
Kawasumi and H.H., unpublished). The expression of Nodal as well as
that of Nodal target genes (Lefty2, Pitx2) is absent in the left LPM
of Cryptic knockout mice, in spite of normal expression of
Nodal at the node (Fig.
3G,K,O) (Yan et al.,
1999). If the indirect signal-relay model is correct, ablation of
Cryptic expression in the node would be expected to prevent
Nodal expression in LPM, given that the secondary signal would not be
generated in or around the node. Alternatively, if the direct transport model
is correct, ablation of Cryptic expression in the node would not be
expected to affect the expression of Nodal in the LPM.
|
). These
results showed that Cryptic in the node is dispensable, with Cryptic
expression in LPM being sufficient, for expression of Nodal in LPM,
providing support for the direct transport model
(Fig. 1B).
|
). These results suggest that, in the absence of this
barrier, Nodal produced in the left LPM is able to travel directly to the
right LPM, where it activates Nodal expression.
Secretion of Nodal at the node
Our results indicated that Nodal produced at the node may travel from the
node to the LPM via the internal route. To examine the fate of Nodal produced
at the node, we generated transgenic mice that harbor a polycistronic
transgene (Fig. 4A) that
encodes 3xMyc-tagged Nodal and ß-galactosidase and is controlled by
the NDE of Nodal (Adachi et al.,
1999). The transgenic embryos exhibited ß-galactosidase
activity only at the node (Fig.
4B), confirming that expression of the transgene is node-specific.
The 3xMyc-Nodal protein appears to retain the functional activity of
native Nodal, given that the transgene rescued the loss of Nodal and
Pitx2 expression in the LPM of
Nodalneo/neo embryos (2/2
and 6/6 embryos, respectively; Fig.
4D,E), which lack Nodal expression in the node and
subsequently that in LPM (Fig.
4C) (Saijoh et al.,
2003). Furthermore, 3xMyc-Nodal was as active as was native
Nodal in a reporter gene assay (Sakuma et
al., 2002) with frog animal caps (C. Tanaka and H.H.,
unpublished).
Given that 3xMyc-Nodal is functional, we first investigated the subcellular localization of this protein by immunofluorescence analysis. Tight junctions and the basement membranes were visualized with antibodies to ZO-1 (Fig. 4I) and laminin (Fig. 4J), respectively, while transgene-expressing cells were detected with an antibody to ß-galactosidase (Fig. 4G,H). The 3xMyc-Nodal protein was detected throughout the cytoplasm of the perinodal crown cells, not only on their apical (ventral) side but also on the lateral and basal sides adjacent to the basement membrane (Fig. 4F,H,I,J).
To examine the secretion of Nodal from the perinodal cells, we prepared immunoenzymatically stained frozen sections from the transgenic embryos (Fig. 5A-D), and ultrathin sections derived therefrom were observed by TEM (Fig. 5E-M). The 3xMyc-Nodal protein was detected specifically in the perinodal crown cells of the transgenic embryos (Fig. 5E-G), whereas no signal was detected in those of non-transgenic embryos (Fig. 5H). The staining was apparent in and around secretory vesicles, which were distributed in both the apical (Fig. 5I) and basolateral (Fig. 5J,K) regions of the perinodal cells. The staining outside the vesicles may have resulted from ultrastructural damage caused during processing of the tissue, most likely during the boiling step (Materials and methods). At higher magnification, signals were detected in the region immediately external to the basolateral (Fig. 5J-M) and apical (Fig. 5I) membranes, possibly reflecting secretion of the 3xMyc-Nodal protein. These observations suggest that Nodal produced at the node is secreted from both the apical and basolateral membranes of the perinodal crown cells. There was no apparent difference in the abundance or localization of 3xMyc-Nodal between the perinodal cells on the left and those on the right (S.O., R.H., H.O. and H.H., unpublished).
Although our results (Fig. 3) suggested that Nodal may travel from the node to LPM, 3xMyc-Nodal was detected only in or immediately external to the perinodal crown cells, with the tagged protein not being apparent in the region between the node and LPM. 3xMyc-Nodal traveling along the internal route, if it exists, must thus be below the limit of detection of this method.
|
) suggests that sulfated GAGs are necessary for the long-range
action of the morphogen Dpp in Drosophila. Although Nodal also
functions over a long distance (Chen and
Schier, 2001; Yamamoto et al.,
2003), the possible role of GAGs in Nodal signaling has been
unknown.
To examine whether GAGs might contribute to long-range signaling by Nodal
from the node to the LPM, we first investigated the distribution of sulfated
GAGs in the E8.0 embryo with antibodies specific for HS or for CS. Both HS and
CS were localized in the basement membrane immediately below the ectoderm and
endoderm layers as well as in the ECM of mesodermal cells
(Fig. 6A,B). The distribution
of HS and CS in the basement membrane was continuous from the node crown cells
to the LPM region. Treatment of the embryo sections with heparitinase
(Fig. 6D,E) or chondroitinase
(Fig. 6G,H), which degrade HS
and CS, respectively, resulted in a marked decrease in immunostaining for the
corresponding GAG. The loss of both GAGs was induced by treatment with both
enzymes (Fig. 6J,K). These
results thus confirmed the specificity of the antibodies. Staining with Alcian
Blue, which detects sulfated GAGs in general, also revealed a distribution
pattern for these molecules similar to those apparent by immunostaining
(Fig. 6C). The Alcian Blue
staining remained but was reduced in intensity after treatment of sections
with either heparitinase or chondroitinase
(Fig. 6F,I), consistent with
the previous finding that approximately equal amounts of HS and CS are present
in the E8.0 embryo (Yip et al.,
2002). Treatment with both enzymes resulted in a loss of Alcian
Blue staining (Fig. 6L),
indicating that HS and CS are the major sulfated GAGs in the E8.0 embryo.
Interaction of Nodal with sulfated GAGs
The specific distribution of sulfated GAGs between the node and the LPM
(Fig. 6) suggested that Nodal
may travel from the node to the LPM through interaction with sulfated GAG
chains. To determine whether Nodal indeed interacts with sulfated GAGs, we
incubated recombinant Nodal in vitro with heparin-sepharose beads and then
subjected the beads to elution with increasing concentrations of NaCl. Most of
the Nodal protein that bound to the beads was eluted by NaCl at concentrations
between 0.15 and 0.90 M, a range that is higher than the physiological salt
concentration, with the peak fraction corresponding to an NaCl concentration
of 0.60 M (Fig. 7A). The
affinity of Nodal for heparin is thus lower than that of typical
heparin-binding proteins, such as heparin-binding epidermal growth factor
(Takazaki et al., 2004), but
it is similar to that of Dpp, which is eluted by NaCl at 0.25 to 0.40 M
(Groppe et al., 1998).
To examine whether Nodal binds to various sulfated GAGs, we subjected the
heparin-sepharose beads with bound Nodal to elution with graded concentrations
of heparin, CS or HS (Fig. 7B).
Most of the bound Nodal protein was eluted by heparin at 10 mg/ml, a
concentration that is 
50 times that of heparin conjugated to the
sepharose beads (10 µl beads contain 20 µg heparin and were
suspended in 100 µl elution buffer, giving a heparin concentration of
0.2 mg/ml). Most of the bound Nodal protein was eluted by CS at 20 mg/ml,
about twice the corresponding value for heparin, suggesting that the affinity
of Nodal for heparin is about twice that for CS. Most of the bound Nodal
protein was not eluted by HS, even at a concentration of 100 mg/ml, indicating
that Nodal does not interact with HS. The failure of HS to induce Nodal
elution was not due to the lower proportion of sulfate groups in HS; the
relative amounts of sulfate groups per unit mass of heparin, CS or HS were
about 3, 1.5 and 1, respectively [estimated by colorimetric quantitation with
Toluidine Blue (S.O. and H.H., unpublished), a metachromatic reagent that
stains sulfate groups of GAGs]. If the affinity is simply proportional to the
amount of sulfate groups, Nodal would have been eluted by HS at 30 mg/ml.
These results thus suggested that Nodal preferentially interacts with heparin
and CS, but not with HS.
|
;
Ullrich and Huber, 2001). By
contrast, p-nitrophenyl ß-D-xylopyranoside (xyloside)
acts as a primer from which GAGs are biosynthesized free from linkage to core
proteins, thus resulting in the production of unlinked sulfated GAGs and naked
proteoglycans (Fig. 9A,H)
(Lugemwa and Esko, 1991).
If sulfated GAGs are required for transport of Nodal from the node to the
LPM, inhibition of the synthesis of these molecules would be expected to
result in failure of the induction of Nodal expression in LPM. We
first determined the optimal concentrations of sodium chlorate and xyloside by
culturing embryos at the headfold stage in the presence of various
concentrations of each reagent until they developed to the six-somite stage.
The optimal concentrations were found to be 15 mM for sodium chlorate and 1 mM
for xyloside. Thus, embryos cultured with 15 mM chlorate remained
morphologically normal, but the amounts of HS and CS immunoreactivity as well
as the intensity of Alcian Blue staining were greatly reduced (4/4 embryos;
Fig. 8K-M) in these embryos
compared with those in control embryos
(Fig. 8B-D). Most of the
embryos cultured with 15 mM chlorate also failed to express Nodal in
LPM, whereas normal asymmetric expression of Nodal was maintained at
the node (Fig. 8E,N,S). The
impaired expression of Nodal in LPM was not due to a reduced
competence of LPM to respond to the Nodal signal, given that chlorate-treated
embryos maintained expression of GDF1 (4/4 embryos) and
Cryptic (4/4 embryos) (Fig.
8F,G,O,P) and because the right LPM of the chlorate-treated
embryos was able to respond to transfection with a Nodal expression
vector (introduced together with a vector for GFP)
(Nakamura et al., 2006) (12/13
embryos; Fig. 8H,I,Q,R).
Culture of embryos with 1 mM xyloside resulted in a marked reduction in the
amount of CS immunoreactivity (4/4 embryos;
Fig. 9C,J) but not in that of
HS immunoreactivity (Fig.
9B,I). Alcian Blue staining also remained but was slightly reduced
in intensity after xyloside treatment (Fig.
9D,K). This preferential effect of xyloside on CS abundance is
consistent with the previous observation that CHO cells cultured with this
reagent secrete xyloside-primed CS rather than HS
(Lugemwa and Esko, 1991). In
the embryos cultured with xyloside, CS would thus be expected to be
synthesized from the xyloside primer and secreted free of core proteins into
the ECM, from which it would be washed away during culture or histological
processing. Whereas asymmetric expression of Nodal was maintained in
the node of these embryos, that in the LPM was not induced in most of them
(Fig. 9E,L,Q). Similar to the
results obtained with chlorate, xyloside did not affect the expression of
Cryptic or GDF1 (Fig.
9F,G,M,N) or the responsiveness of LPM to the Nodal signal (10/11
embryos; Fig. 9O,P).
|
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This notion gained support by the detection of
nodal vesicular parcels (Tanaka et al.,
2005). Nodal secreted from the perinodal cells toward the node
cavity would be expected to be transported by the flow and might signal
through the endoderm. However, we have now shown that the endoderm does not
express Cryptic and is not responsive to the Nodal signal, making it
unlikely that Nodal transported by nodal flow signals through the endoderm. A
more likely scenario is that Nodal secreted basolaterally from the perinodal
cells is responsible for induction of Nodal expression in LPM.
In the chicken embryo, the L-R signal generated at the node is transmitted
to the LPM by signal relay. Thus, Sonic hedgehog produced at the node induces
expression of a bone morphogenetic protein (BMP) antagonist, Caronte, which in
turn activates Nodal expression in LPM
(Yokouchi et al., 1999).
However, we have now shown that Cryptic, a Nodal co-receptor essential for
Nodal signaling, is dispensable in the node and is required only in the LPM
for induction of Nodal expression in LPM. Cryptic is the
only EGF-CFC family gene expressed in the node of E8.0 mouse embryo;
Cripto, another member of this family, is not expressed there at this
stage (see Fig. S2 in the supplementary material). These observations suggest
that the Nodal signal is not relayed between the node and LPM but rather is
directly transported. This notion is consistent with previous observations
showing both that Nodal is able to act over a long distance
(Chen and Schier, 2001;
Yamamoto et al., 2003) and
that asymmetric Nodal expression in LPM is governed by two enhancers
(ASE and LSE), both of which are responsive to the Nodal signal
(Saijoh et al., 2000;
Saijoh et al., 2005).
How does bilateral Nodal expression in the node result in asymmetric Nodal expression in LPM?
Nodal is asymmetrically expressed exclusively in left LPM, with
its bilateral expression at the node showing only a subtle L-R asymmetry. How
is such a robust asymmetry generated in the LPM? If Nodal is transported from
the node to LPM, is it transported preferentially toward the left side?
Although Nodal expression in the node exhibits a subtle asymmetry,
with the level of expression on the left side being slightly higher than that
on the right side, this subtle asymmetry does not appear to be essential for
the robust asymmetry in LPM, because Nodal expression in left LPM is
maintained in the transgenic embryos that express Nodal symmetrically
at the node (Norris et al.,
2002; Saijoh et al.,
2003). Therefore, other genes expressed in the node with subtle
asymmetry, such as LPlunc1 and Cerberus-like 2, may play a
role in this process (Hou et al.,
2004; Marques et al.,
2004; Pearce et al.,
1999).
Alternatively, although Nodal mRNA is distributed bilaterally at
the node, nodal flow may lead to the biased production of more active Nodal
protein on the left side of the node. For instance, post-translational
cleavage or secretion of cleaved products may occur more efficiently on the
left side, in response to nodal flow. A Ca2+ signal that exhibits
asymmetry between the two sides of the node, being stronger on the left
(McGrath et al., 2003), may
regulate protein secretion. We did not detect a difference in the number of
secretory vesicles containing Nodal between the two sides of the node
(Fig. 5). However, such a
difference may not necessarily be large, given that a small difference
generated at the node can be autonomously converted to a robust asymmetry in
LPM by a self-enhancement and lateral inhibition mechanism
(Nakamura et al., 2006).
|
). Nodal
was eluted from heparin-sepharose beads by NaCl most effectively at 0.45 to
0.60 M, suggesting that it possesses an intermediate affinity for heparin.
Such an intermediate affinity might be expected to be ideal for transport of a
protein over a long distance via sulfated GAGs (a protein with a higher
affinity would become trapped by the sulfated GAGs).
The physical interaction of Nodal with sulfated GAGs, the specific
localization of sulfated GAGs to the basement membrane-like structure between
the node and the LPM, and the pronounced effects of inhibition of sulfated GAG
synthesis observed in the present study all are consistent with a requirement
for sulfated GAG chains in the efficient transport of Nodal from the node to
the LPM. In Drosophila, Dpp is not able to move across cells
deficient in HS biosynthesis (Belenkaya et
al., 2004). Similarly, in the absence of sulfated GAGs, Nodal may
not be transported efficiently or may become unstable. Our results suggest
that CS, rather than HS, plays an important role in the long-range action of
Nodal, given that Nodal interacts with CS but not with HS and that
Nodal expression in LPM was impaired as a result of inhibition of CS
proteoglycan biosynthesis by xyloside.
|
).
Xenopus embryos treated with xyloside thus failed to undergo heart
looping, a typical L-R patterning defect. Although the molecular mechanism of
this effect of xyloside was not investigated, it is likely that asymmetric
expression of Xnr-1 in LPM was lost in such embryos. Furthermore, the
crucial period for the xyloside effect was between stages 12 and 15, which is
immediately before the onset of asymmetric expression of Xnr-1 in LPM
and probably corresponds to the stage of the mouse embryos in our xyloside
experiments. Sulfated GAGs may therefore play a role in Nodal signaling in the
frog embryo similar to that demonstrated in the mouse embryo, although
xyloside appears to interfere with the synthesis of HS rather than with that
of CS in Xenopus.
To date, more than ten genes have been identified in the mouse genome that
encode enzymes involved in CS biosynthesis
(Silbert and Sugumaran, 2002).
Two of these genes, those encoding chondroitin 6-O-sulfotransferases
2 and 3, have been studied by generation of mutant mice, but neither of the
mutants exhibited L-R patterning defects
(Uchimura et al., 2004;
Uchimura et al., 2002),
possibly as a result of compensation by other chondroitin
6-O-sulfotransferase genes. Further loss-of-function analysis of CS
biosynthetic enzymes is thus required to establish the precise role of CS in
Nodal signaling.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/21/3893/DC1
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ACKNOWLEDGMENTS |
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Present address: Departments of Medicine and Genetics & Development,
Columbia University College of Physicians and Surgeons, Herbert Irving
Comprehensive Cancer Center, 1130 St Nicholas Ave, Room 217B, New York, NY
10032, USA
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REFERENCES |
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